Retrospective Theses and Dissertations Iowa State University Capstones, Theses andDissertations

2003

The role of Rev-SR protein interactions in theregulation of equine infectious anemia virusreplicationGregory Saang ParkIowa State University

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Recommended CitationPark, Gregory Saang, "The role of Rev-SR protein interactions in the regulation of equine infectious anemia virus replication " (2003).Retrospective Theses and Dissertations. 611.https://lib.dr.iastate.edu/rtd/611

The role of Rev-SR protein interactions in the regulation of equine infectious anemia virus replication

by

Gregory Saang Park

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Genetics

Program of Study Committee: Susan L. Carpenter, Major Professor

Norman F. Cheville W. Allen Miller Chris K. Tuggle Daniel F.Voytas

Iowa State University

Ames, Iowa

2003

Copyright © Gregory Saang Park, 2003. All rights reserved.

UMI Number: 3085936

Copyright 2003 by

Park, Gregory Saang

All rights reserved.

®

UMI UMI Microform 3085936

Copyright 2003 by ProQuest Information and Learning Company.

All rights reserved. This microform edition is protected against

unauthorized copying under Title 17, United States Code.

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ii

Graduate College Iowa State University

This is to certify that the doctoral dissertation of

Gregory Saang Park

has met the dissertation requirements of Iowa State University

Maf r Professor

For t Major Program

Signature was redacted for privacy.

Signature was redacted for privacy.

iii

Dedication

For my father, Dr. Yong Ho Park

For my mother, Sharon Park

You gave me life and shaped my mind.

You did the best with what you had to work with.

Thank you.

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS vi

ABSTRACT vii

CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 1 Introduction 2 Overall Goal 19 References 19

CHAPTER 2. FUNCTIONAL CHARACTERIZATION OF TAT ACTIVITY FROM EIAV ALTERNATIVELY SPLICED MESSENGER RNAS 34

Abstract 34 Introduction 35 Materials and Methods 37 Results and Discussion 38 References 41

CHAPTER 3. SF2/ASF INHIBITS EQUINE INFECTIOUS ANEMIA VIRUS REV ACTIVITY AND VIRAL REPLICATION 53

Abstract 53 Introduction 54 Materials and Methods 56 Results 62 Discussion 66 References 70

CHAPTER 4. GENERAL CONCLUSION 91 Alternatively Spliced Tat Transcripts 91 SF2/ASF Inhibits EIAV Rev-mediated Nuclear Export and EIAV Replication 92 Future Studies 93 References 95

APPENDIX A. THE PURIFICATION OF EIAV REV 97 Introduction 97 Methods and Results 99 Acknowledgements 105 References 105

V

APPENDIX B. BINDING OF EQUINE INFECTIOUS ANEMIA VIRUS REV TO AN EXON SPLICING ENHANCER MEDIATES ALTERNATIVE SPLICING AND NUCLEAR EXPORT OF VIRAL MRNAS 110

Abstract 110 Introduction 111 Materials and Methods 113 Results 118 Discussion 123 Acknowledgements 127 References 127

VI

ACKNOWLEDGEMENTS

First of all, I thank my major professor Dr. Susan L. Carpenter for the many years of

time, effort, and money that she has sunk into my education and development to become a

scientist. It is comforting to know that almost 1/5* of my current lifespan has been spent in

an office and laboratory under the guidance of a truly good scientist. In addition, she has

provided me with her friendship and a mutual interest in outside activities, which proved

invaluable during my years in Iowa.

I thank the current and former members of the Carpenter laboratory for their insight,

discussions, knowledge, technical expertise, and personal friendships: Michael Belshan,

Prasith Baccam, Pam Bruelman, Amanda Johnson, Yuxing Li, Sean Murphy, Mariah Porter,

Brett Sponseller, Robert J. Thompson, Nick Wills, and Wendy Wood. I especially thank

Mike and Yvonne Wannemuehler, who are dear to me. You have been so kind, you have

shared so much, and you have helped me when I was in need.

I must thank my many friends who have offered advice and played such an important

role in my life. You know who you are. I cannot name you all. There would not be enough

room. Special thanks to a dear friend, Carissa Steelman.

Finally, I thank my program of study committee members:

Norman Cheville, W. Allen Miller, Chris Tuggle, and Daniel Voytas.

vii

ABSTRACT

Equine infectious anemia virus (EIAV) is a member of the lentivirus subfamily of

retroviruses that produces a variable clinical disease course characterized as acute, chronic,

and inapparent. The clinical signs can vary according to the stage of disease, and generally

correlate with levels of virus replication. As with other retroviruses, EIAV utilizes both

RNA and proteins to produce alternatively spliced transcripts required for virus replication.

EIAV encodes a protein called Rev, which functions by binding unspliced and singly spliced

viral mRNAs in the nucleus at a sequence called the Rev responsive element (RRE) and

exporting them into the cytoplasm. Rev is absolutely required for virus replication, and

factors that inhibit Rev function would be expected to inhibit virus replication. EIAV Rev is

encoded in exons 3 and 4 of a bicistronic, four-exon mRNA, which also encodes the protein

Tat in exons 1 and 2. The presence of Rev results in the expression of an alternatively

spliced viral mRNA that differs from the four-exon mRNA by lacking exon 3. Exon 3

contains cis-acting sequences that function as both an exon splicing enhancer (ESE) and a

RRE. ESEs bind cellular SR proteins to assist in the recognition and inclusion of exons.

Therefore, the EIAV ESE/RRE sequences bind both SR proteins and Rev. The goal of this

research is to characterize the interactions between EIAV and cellular SR proteins that

modulate virus replication. I first show that Rev-mediated alternative splicing of exon 3 is

not a mechanism to up-regulate Tat activity. I demonstrate that SF2/ASF inhibits Rev-

mediated nuclear export activity and EIAV replication in vitro. I show that the RNA binding

domain of SF2/ASF is necessary and sufficient for the inhibition of Rev nuclear export

activity and EIAV replication. Further, the inhibition of both Rev activity and virus

viii

replication correlated with the SR protein RNA binding specificity. These results suggest

that SR proteins and Rev compete for binding viral RNAs at the ESE/RRE. Therefore,

factors that modulate intracellular concentrations of SR proteins may play a role in regulating

Rev nuclear export activity and EIAV replication.

1

CHAPTER 1. GENERAL INTRODUCTION

Dissertation Organization

This dissertation biologically characterizes the interactions of virus and host cell

factors in equine infectious anemia virus (EIAV) alternative splicing. This dissertation is in

the alternative format and has four chapters and two appendices. The first chapter provides a

general background of retroviruses, the importance of splicing in retroviral gene expression,

and introduces the retrovirus EIAV. There is a description of splicing and the SR protein

family of splicing regulators, with an emphasis on the SR protein SF2/ASF. Finally, there is

a detailed description of SF2/ASF and EIAV Rev interactions with EIA viral RNAs. Chapter

2 is a paper to be submitted for publication in the journal Virus Genes that is a functional

characterization of the monocistronic Tat transcripts of EIAV. This work was done in

collaboration with Michael Belshan and Susan Schommer. Susan Schommer and Michael

performed the cloning of the EIAV cDNAs. The monocistronic Tat transcript is an

alternatively spliced variant of a bicistronic transcript that encodes Tat and Rev. The

presented data indicates that Tat activity from either monocistronic or bicistronic viral

mRNAs was not significantly different, suggesting that RNA of the downstream rev gene

does not affect translation initiation at the CUG start codon of the upstream tat gene.

Chapter 3 is a paper to be submitted for publication in the journal Molecular and Cellular

Biology that characterizes SF2/ASF inhibition of both EIAV Rev activity and EIAV

replication. All of the work in this manuscript was performed by myself. The RNA binding

domain of SF2/ASF was found to be necessary and sufficient for the inhibition of Rev

activity and virus replication. The results suggest that SF2/ASF competes with Rev for

2

binding viral RNAs, inhibiting Rev activity, and subsequently, EIAV replication. Chapter 4

includes the general conclusions of my dissertation research as well as my recommendations

for future studies. Appendix A is a detailed description of the methods used and the progress

made towards purifying the protein EIAV Rev, which will be used in RNA binding

competition studies with SF2/ASF, and in mapping the Rev RNA binding domain. Appendix

B is a paper published in the journal Molecular and Cellular Biology by Michael Belshan

that is a molecular characterization of Rev-mediated alternative splicing. Whereas the

majority of the paper was written and performed by Michael Belshan, I performed the protein

purification of Rev and the in vitro assays in which SF2/ASF inhibited Rev-mediated nuclear

export. The purification of Rev was essential to much of the work in the paper. The protein

was used in the in vitro binding assays for the identification of the RRE, and in the in vitro

splicing assays to show that Rev inhibits exon 3 splicing.

Introduction

Retroviruses are a family of enveloped, ssRNA viruses, whose hallmark is the use of

a viral encoded RNA-dependent DNA polymerase, or reverse transcriptase, to produce a

linear, dsDNA copy from a ssRNA genome (reviewed in 19). In retroviral replication, a

retrovirus attaches to a host-cell, fuses with the lipid bi-layer, and the viral core enters the

cytoplasm (Figure 1). The viral core goes through a process of uncoating and the packaged

reverse transcriptase creates the intermediate dsDNA genome, which then translocates into

the nucleus and integrates into the host-cell genome. From the integrated viral DNA copy

called the provirus, transcription at the 5'-long terminal repeat (LTR) produces full-length

transcripts that are the source of all viral mRNAs necessary for replication. All retroviruses

3

Fusion

Reverse Transcription

Uncoating Attachment Nuclear Translocation

Proteolytic maturation BZ> •—

Progeny RNA

Translation

Assembly

Budding

Figure 1. General retroviral replication cycle. After attachment, the retroviral and cellular

membranes fuse, releasing the core into the cytoplasm. The core goes through a process of

uncoating, and the ssRNA genome is reverse transcribed into dsDNA with the packaged

reverse transcriptase. The gray/black/white boxes indicate the U3 (gray), R (black), and U5

(white) sequences that make up the long terminal repeats (LTRs) of the retrovirus. The

dsDNA copy of the viral genome translocates into the nucleus, integrates into the host cell

genome, and transcription from the single viral promoter in the 5' LTR produces unspliced

transcripts. The unspliced transcripts encode gag and pol and serve as new virion genomes.

For retroviral replication, some unspliced transcripts must be spliced to produce the singly

spliced transcripts encoding env. Virion proteins and genomes are assembled at the cell

membrane and new infectious viruses are produced by budding and proteolytic maturation.

Figure adapted from Coffin et al. 1997 (19).

produce mRNAs that encode three major coding regions for the polyproteins gag, pol, and

env. The group-specific antigen gene, gag, encodes the major internal core structural

proteins: matrix, capsid, and nucleocapsid. The pol gene encodes minimally the reverse

4

transcriptase and integrase enzymes, and env encodes the envelope glycoproteins made up of

surface and transmembrane components, which have roles in cell attachment and virus entry.

In addition, all retroviruses encode a protease in the gag and/or pol ORFs, which plays a role

in virion maturation. Because retroviruses have a limited genome size and a single promoter,

they utilize the host-cell splicing machinery that acts on unspliced viral transcripts to produce

multiply spliced transcripts that are necessary for replication.

Figure 2. Molony murine leukemia virus genome and transcripts. The boxes indicate the open

reading frames, the horizontal dark lines indicate exon sequences, and the diagonal lines

indicate the spliced out RNA sequence.

For some retroviruses, such as Moloney murine leukemia virus, inefficient splicing of

the unspliced transcript produces a second transcript that encodes the env (Figure 2). For

other retroviruses, virally encoded proteins regulate RNA synthesis and expression. For

example, the Lentiviridae subfamily of retroviruses encodes two proteins Tat and Rev

(reviewed in 26,85). Tat functions by entering the nucleus and binding to a bulged, stem-

loop structure found at the 5' terminus of all viral mRNAs called the Tat activation region

(TAR) (22,28,42,74,75). Tat binds TAR in association with cyclin T1 (91) and cyclin-

dependent kinase 9 (Cdk9) (7). In this complex called P-TEFb, Cdk9 phosphorylates the C-

LTR

env

5

terminal domain of RNA polymerase H (RNAPII) (59,94,98), which shifts transcription from

initiation to elongation. Therefore, Tat up-regulates transcription.

Figure 3. Mechanism of Rev function. Rev enters the nucleus, binds singly spliced and

unspliced viral mRNAs, multimerizes, and exports the mRNAs into the cytoplasm. The

singly spliced and unspliced viral mRNAs encode the structural proteins of the virus and the

unspliced mRNA serves as the genomes for new virions. The cytoplasmic expression of the

structural proteins is dependent on Rev, and therefore, Rev is absolutely required for virus

replication.

A second lentiviral regulatory protein is Rev. The prototypical and most-

characterized Rev is human immunodeficiency virus (HIV) Rev (reviewed in 26,48,72). Rev

functions by entering the nucleus, binding singly and unspliced viral mRNAs at a sequence

in the env called the Rev responsive element (RRE) (20,97), multimerizing (71,96), and

exporting the mRNAs into the cytoplasm through the exportin 1 (Crml) nuclear export

pathway (Figure 3) (29,30). Because Rev is necessary for the cytoplasmic expression of the

unspliced and incompletely spliced viral mRNAs, Rev is absolutely required for virus

replication.

Provirus

•AAAA pre mRNA

AAA

AAAA exportin 1

Structural Proteins Structural Proteins and

Progeny RNA genome importin (3

An in vitro assay has been developed to measure Rev nuclear export activity (4,5,44),

which uses a chloramphenicol acetyltransferase (CAT) reporter plasmid (Figure 4). When

transacted into cells, the CAT-based Rev reporter expresses mRNAs that, from 5' to 3',

consist of a splice donor, a CAT gene, a RRE, and a splice acceptor. Therefore, the CAT

gene and RRE are within RNA sequence that is recognized as an intron. In the absence of

Rev, the CAT gene and RRE are spliced out of the transcript and no CAT is expressed.

However, if the reporter is in the presence of Rev, Rev binds the RRE and exports the

unspliced reporter transcript into the cytoplasm, and CAT is expressed. CAT can then be

assayed as a quantification of Rev nuclear export activity.

SD SA r — i l - . ; I

- - - • CMV -pi CAT —! HREi—r

+

+

= —| CAT —

Figure 4. In vitro Rev nuclear export reporter. The Rev reporter plasmid produces transcripts

that contain a CAT gene and a RRE flanked by a splice donor (SD) and a splice acceptor

(SA). In the absence of Rev, the CAT gene and RRE are spliced out of mRNAs, whereas in

the presence of Rev, Rev binds the RRE, exports reporter transcripts into the cytoplasm, and

CAT is expressed.

Equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that encodes both Rev and Tat

proteins. EIAV is closely related to caprine arthritis-encephalitis virus as well as the human,

simian, and feline immunodeficiency viruses (HIV-1 and -2, SIV, FIV). Infection with

7

EIAV produces a persistent, lifelong infection in horses and other members of the family

Equidae (reviewed in 77,78). Most EIAV-infected horses show little to no sign of infection

(45), but the disease course may vary to include periods of acute, chronic, and/or inapparent

disease. Clinical signs include cycles of high fever, thrombocytopenia and/or anemia, and

each febrile episode is associated with viremia. Interestingly, there is no single immune

parameter that correlates to the control of virus replication or the clinical disease in EIAV

infection (36-38). EIAV is transmitted through blood or blood products, most commonly by

the large biting insects of the Tabanidae family (horseflies); another common method of

transmission is through the use of contaminated blood transfusions, needles, or surgical

equipment. EIAV is tropic to cells of the monocyte/macrophage lineage, with the majority of

virus replication occurring in the tissue macrophages (70,79), and the highest titers of virus

found in the serum, liver, spleen, bone marrow, lung and kidney (79).

There are five major transcripts that have been identified in EIAV replication (68). A

fully spliced, four-exon, bicistronic mRNA encodes both Tat and Rev (mRNAl, Figure 5)

(67,81). Tat is translated from exons 1 and 2, and Rev is translated from exons 3 and 4

(23,24,67,69,81), though the known functional domains of Rev are wholly encoded in exon 4

(31,40,60). In the absence of Rev, only the bicistronic mRNA is expressed in the cytoplasm

(62,76), and thus, Rev mediates the cytoplasmic expression of the other EIAV mRNAs. One

of the four mRNAs is an alternatively spliced mRNA identical to the fully spliced, four-exon

mRNA, but lacking rev exon 1 and encoding only Tat (mRNA 2, Figure 5) (62). As with

other lentiviral Rev's, EIAV Rev binds and exports the unspliced and singly spliced viral

mRNAs, which encode the structural genes (mRNA 4 and mRNA 5, Figure 5).

8

po! f757!

Til gp9Q 1 gp45 M gag

mRNA

I ttm

gp9Q gp45

gag

Protein

Tat, Rev

Tat

Ttm

Env

Gag, Pol

Figure 5. The EIAV genome, major transcripts, and the proteins encoded. Messenger RNA I

encodes the regulatory proteins Tat and Rev. In the presence of Rev, mRNA2 is produced,

which is identical to mRNAl, but lacks rev exon I. Messenger RNA3 encodes a truncated

transmembrane protein (Ttm), which has not been well characterized. Rev also mediates the

cytoplasmic expression of the mRNAs encoding structural proteins and serving as the

progeny genome (mRNA4 and mRNA5).

EIAV Rev is a 165 amino acid protein that is functionally homologous to HIV Rev,

yet shares little amino acid homology. EIAV Rev is not as well characterized as HIV Rev,

but some functional domains have been identified, including the nuclear localization signal

(NLS)(amino acids 160-165) (65) and the nuclear export signal (NES) (amino acids 32-55)

(31). The RNA binding domain (RED) has not been mapped, but recent studies suggested

that a region of the protein that mediates alternative splicing (amino acids 75-127) may be

the RNA binding domain (65). Finally, some studies suggest that EIAV Rev also

multimerizes, but the domain has not been mapped (88). Interestingly, the EIAV functional

domains differ in their structural organization compared to HIV Rev (Figure 6). The residues

of the HIV Rev RBD/NLS are located toward the amino end of the protein and are flanked

9

by the residues of the multimerization domain, and the residues of the HIV Rev NES are

located toward the carboxy end.

EIAV Rev Alternative Splicing/

NES RNA binding? NLS

NLS/RBD

HIV Rev i r " 1

NES

t t MULTIMERIZATION

Figure 6. The domain organization of both EIAV Rev and HIV Rev. Though they are

functionally homologous, the domain organization is different between EIAV Rev and HIV

Rev. Both proteins contain nuclear export signals (NES) and nuclear localization signals

(NLS), though the HIV Rev NLS is also the RNA binding domain (RBD). In contrast, the

RBD of EIAV Rev has not been well characterized, but may be the identified alternative

splicing domain. Finally, the multimerization domain of HIV Rev flanks the NLS/RBD,

whereas EIAV Rev multimerization has not been well characterized.

Pre-mRNA Splicing

There is growing evidence that many of the major processes within the nucleus

including transcription, splicing, and nuclear export, are not separate, but intricately

connected (reviewed in 6,25,33). Splicing is a post-transcriptional modification performed

on pre-messenger RNA (pre-mRNA) to remove introns and join segments of RNA as exons

(reviewed in 33,41,52). The splicing reaction is carried out by the splicing machinery, and

involves many interactions between a number of cis-acting pre-mRNA sequences and a

larger number of trans-acting factors.

Three cis-RNA sequences are necessary in splicing. These are the 5' splice site, the

branch point, and the 3' splice site. The 5' splice site is also called the splice donor, and it has

the consensus RNA sequence ^AGjGUPuAGLP6, where the line between the AG and GUPu

marks the border between the upstream exon and downstream intron, and the Pu represents

either purine nucleotide (G or A). The branch point is downstream of the 5' splice site and

has a consensus RNA sequence CUPuAPy, where Py denotes either C or U, and also is the

beginning of a polypyrimidine tract. The 3' splice site is also called the splice acceptor, and

it has the consensus RNA sequence "4NPyAG|PuN+2. The line between the AG and PuN

marks the border between the upstream intron and the downstream exon, and the N

represents any RNA nucleotide. Thus, the 5' splice donor defines the downstream boundary

of one exon, and the 3' splice acceptor defines the upstream border of another exon.

The trans-acting splicing machinery includes, amongst other proteins, a multi-protein,

catalytic RNA-protein complex called the spliceosome. The spliceosome is made up of a

large number of proteins that are divided into two groups: the small nuclear

ribonucleoprotein complexes (snRNPs), which are RNA-protein complexes, and the non-

snRNPs, which are also called general splicing factors (GSFs). Spliceosome assembly is

initiated through snRNP components at the cis-KNA sequences. During assembly, major

complex formations are made by an ordered interaction of other snRNPs (Figure 7). In the

commitment or early (E) complex, the pre-mRNA is bound at the 5' splice site by the U1

snRNP, at the branch point by splicing factor 1 and 65 kDa subunit of the heterodimeric

factor U2AF, and at the 3' splice site by the 35 kDa subunit of U2AF. It is thought that the E

complex defines exons such that the formation of the E complex commits the pre-mRNA to a

particular splicing choice. Complex A is formed when the U2 snRNP displaces splicing

factor 1 and U2AF. Complex B1 and then Complex B2, the mature spliceosome, follow

Complex A when the U5/U4-U6 tri-snRNP and a number of GSFs interact with the pre-

11

mRNA. During the interaction, the RNA component of U6 base pairs with the 5' splice

donor, and the protein component of U6 associates with the U2 snRNP. The U5 snRNP and

GSFs remain associated with the spliceosome, and both the U4 and U1 snRNPs are released.

Many of the GSFs involved in spliceosome assembly belong to a family of proteins called

the SR proteins.

FUT

U4

(BP)

UI

I U2AF/ 1

ISF1 / s s t

U2AF 3

U1

I I til 15'E7i l?y

pa AGI

ACAGA" U6

ÀCÂGÂ' U6

~U2~]

Early (E) Complex

t Complex A

• Complex B2

Figure 7. Spliceosomal assembly. The commitment complex, or E complex, forms when the

pre-mRNA is bound by the Ul snRNP at the 5' splice donor, by both splicing factor 1 (SF1)

and the 65 kDa subunit of U2AF at the branch point (BP), and by the 35 kDa subunit of

U2AF at the 3' splice acceptor. The U2 snRNP binds the branch point and displaces SF1 and

U2AF to form Complex A. The mature spliceosome, Complex B2, is formed when the

U5/U4-U6 tri-snRNP interacts with the pre-mRNA, releasing the U4 and Ul snRNPs. The

figure was adapted from Murray and Jarrell, 1999 (66).

12

SR Proteins

SR proteins are a family of proteins that are involved in the process of splicing during

pre-mRNA processing, and are also involved in other stages of gene expression including

transcription, 5' capping, polyadenylation, and nucleocytoplasmic transport (reviewed in

8,9,14,33,58,84). SR proteins function in both exon-independent and exon-dependent

splicing (43). In exon-independent splicing, SR proteins assist in protein-protein interactions

during the splicing reaction, but they do not directly interact with the pre-mRNA. In exon-

dependent splicing, SR proteins bind pre-mRNA to aid splice site recognition by the

components of the splicing machinery. The necessity for exon-dependent splicing is

typically conditional on the strength of the splice site, which is judged by the splice site's

conformity to a consensus sequence.

Members of the SR protein family are generally characterized by the presence of one

or two amino-terminal RNA recognition motifs (RRMs) and a carboxy-terminal RS domain.

The RRM conforms to a consensus RNP-type RNA binding domain, whereas the RS domain

consists of a number of arginine-serine dipeptide repeats. A second defining characteristic of

SR proteins is the ability to activate splicing in a SR protein-depleted in vitro splicing assay.

To date, 10 human SR proteins have been identified (SRp20, SF2/ASF, SC35, SRp30c, 9G8,

SRp40, SRp46, SRp55, SRp75, p54), and numerous highly conserved homologs, orthologs,

and other SR family members have been identified in a wide variety of species of both plants

and animals including: Arabidopsis thaliana, Chironomas tentons, Caenorhabditis elegans,

Mus musculus, and Drosophila melanogaster (1,2,13,56,57). The two prototypical SR

proteins that separate the one and two RRM-containing protein members are SC35 and

SF2/ASF (99).

The SR Protein Functional Domains

The main function of the SR protein RRM is in determining substrate-specificity by

binding to RNA (17,46,55,63,64,84,87). The consensus RNA binding sequence of specific

SR protein RRMs has been determined using a procedure called SELEX (sequential

evolution of ligands by exponential enrichment) (86). The results of SELEX analyses shows

that each RRM can bind a number of different RNA sequences, but there are specific RNA

sequences that distinct SR proteins will bind. In addition, SR proteins that have two RRMs

require both to bind a specific RNA sequence (99). Therefore, sequence-specific RNA

binding depends not only on the identity of the RRM, but also on the number of RRMs (83).

The other functional domain of the SR proteins is the RS domain. The major

functional role of the RS domain is in mediating the protein-protein interactions among SR

proteins and the components of the splicing machinery (49,92). These interactions include,

but are not limited to, phosphorylation, nuclear localization, and direct interactions with other

SR proteins as well as other splicing proteins. RS domains may also have some minor role in

determining substrate specificity, but they contribute little to RNA binding (17,63,83). RS

domains are somewhat conserved at the amino acid level, and for some SR proteins, are

functionally interchangeable (17,90). Phosphorylation and dephosphorylation of the RS

domain modulates the activity of SR proteins, and phosphorylation is mediated by the

interaction of the RS domain with two SR protein kinases (SRPK1 and SRPK2) (50). The

phosphorylation state of the RS domain also affects SR protein intracellular movement and

subcellular localization. RS domains function as sufficient nuclear localization signals, and

interact with two importin g family nuclear import proteins called transportin-SR (TRN-SR)

and transportin-SR2 (TRN-SR2) (47,53). For some SR proteins, the RS domain also confers

an ability of the SR protein to shuttle from the nucleus back into the cytoplasm (11,16). In

addition, RS domains are required for SR protein movement between the nuclear speckles

(storage sites) and sites of active transcription (50,64).

SR Protein Regulated Alternative Splicing

SR proteins play a role in both constitutive and alternative splicing (reviewed in

14,35,61). Constitutive splicing is simply the removal of an intron between two exons,

whereas alternative splicing is the differential use of multiple splice sites in a pre-mRNA to

construct different mRNAs consisting of various exons. The four most common modes of

alternative splicing are: exon exclusion/inclusion, use of alternative 3' splice acceptors, use

of alternative 5' splice donors, and mutually exclusive exons (Figure 8). SR proteins

function in a parallel and concentration-dependent manner in splicing. For example, a

particular SR protein may be sufficient but not necessary to assist in the splicing of a

substrate, as it can be replaced by a different SR protein. In addition, SR proteins may assist

in exon inclusion during alternative splicing at a certain concentration, but an increase or

decrease in that concentration may change the choice of splice site utilization and result in a

different mRNA. SR protein expression varies in a number of tissues and cellular activation

states (39,54,95), and it has been shown that alternative splicing occurs in a tissue-specific

manner (80).

It is generally agreed that SR proteins function in alternative splicing by binding to

RNA sequences within pre-mRNAs and assisting in the recognition of non-consensus splice

sites, which leads to the inclusion of exons (reviewed in 8,14,84). In particular, SR proteins

are involved during E complex formation, which is the complex that commits the pre-mRNA

15

to a splicing pattern. For example, the SR proteins SF2/ASF and SC35 interact with the Ul-

70kDa protein subunit of the Ul snRNP, and assist in stabilizing the Ul snRNP interaction

with the 5' splice donor (12,27,49,93). In addition, SR proteins can bind U2AF and assist in

the recognition of 3' splice acceptor (92).

Exon exclusion/inclusion

Alternative 3' splice acceptors —[

Alternative 5' splice donors

Mutually exclusive exons

Figure 8. Four common modes of alternative splicing. Each mode shows two possible

splicing choices, and are represented by either the top diagonal lines or bottom diagonal lines.

For example, in exon exclusion/inclusion, the mRNA may be spliced to include the exon (top

diagonal lines), or may be spliced to exclude the exon (bottome diagonal lines). The boxes

indicate the exon sequences, the horizontal lines indicate the intron sequences, and the

diagonal lines indicate the spliced intron. Adapted from Cartegni et al., 2002 (14).

In alternative splicing, the prevalent theory of exon exclusion/inclusion is called exon

definition, which proposes that the exon, not the intron, is the unit recognized by the splicing

machinery (73). Thus, there is communication between the 3' splice acceptor and the 5'

splice donor that flank an exon in order for the spliceosome to recognize the exon and

include it during pre-mRNA processing (73). Because SR proteins can interact with both the

Ul-70k and U2AF proteins simultaneously (92), it is thought that SR proteins assist in the

recognition of the splice sites as well as bridge the interactions between the proteins at the

upstream 3' and downstream 5' splice sites. Indeed, many intraexonic RNA binding sites

have been discovered, and have an active role in regulating alternative splicing.

In addition to the 5' splice donor, branch point, and 3' splice acceptor, there are two

groups of CLV-RNA elements that also modulate spicing. Those that mediate the inclusion of

an exon during constitutive or alternative splicing are referred to as exon splicing enhancers

(ESEs). Those that mediate the exclusion of an exon are referred to as exon splicing

silencers (ESSs) (3,82). SR proteins function by binding ESEs and recruiting the splicing

machinery to non-consensus (weak) splicing signals through RS domain-mediated protein-

protein interactions. SR proteins may also function by binding RNA and antagonizing the

action of nearby ESSs. Investigation has identified a large number ESEs that are purine-rich

and intraexonic, but ESEs are not strictly purine-rich (15,21).

The SR Protein SF2/ASF

One particular SR protein that is extensively studied in the process of splicing is the

protein Splicing Factor 2/Alternative Splicing Factor (SF2/ASF). SF2/ASF is also referred to

as ASF/SF2 (32,51). SF2/ASF is highly conserved among mammals, and numerous

homologs and orthologs have been discovered in other species including birds, plants,

worms, and insects (1,2,13,56,57,95). For example, between humans and mice, the amino

acid sequence of SF2/ASF is 100% identical. SF2/ASF is also an essential gene for cell

viability (89). Some of the major effects of SF2/ASF in splicing are to (i) assist the Ul

snRNP bind the 5' splice donor, (ii) to assist the U2AF bind the 3' splice acceptor, and (iii) to

play a role in the first part of the splicing reaction (12,49,92).

17

SF2/ASF

97 107 197 198 248 -W

RRM1 RRM2 m Figure 9. Domain organization of SF2/ASF. SR proteins consist of one or two amino-

terminal RNA recognition motifs (RRMs) and a carboxy-terminal RS domain made-up of a

number of arginine-serine dipeptide repeats. The RRMs function in determining substrate

specificity by binding to RNA and the RS domain mediates the protein-protein interactions of

the SR protein. SF2/ASF contains two RRMs and a RS domain. The numbers above the

figure show the amino acids that define the domain. Between the two RRMs is a glycine-rich

hinge.

SF2/ASF is a 248 amino acid protein with an apparent molecular weight of 33 kDa

and a predicted molecular weight of 27.7 kDa. The functional domains of SF2/ASF include

two amino-terminal RRMs and a carboxy-terminal RS domain (Figure 9) (10,99). Amino

acids 1-97 constitute RRMl, amino acids 107-197 constitute RRM2, and amino acids 198-

248 constitute the RS domain. Between the two RRMs is a glycine rich region (amino acids

98-106) that is suggested to serve as a hinge between the two RRMs. SF2/ASF is considered

the prototype of a class of SR proteins that have two RRMs (99).

Using various SELEX protocols, three different consensus RNA binding motifs have

been identified for SF2/ASF: RG A AG A AC, AGGACAGAGC, and SRSASGA (R=G/A and

S= C/G) (55,83). SELEX analyses with only RRMl of SF2/ASF resulted in the consensus

RNA sequence ACGCGCA. ESE sequences that conform to the SF2/ASF RNA binding

motifs and specifically bind wild-type SF2/ASF also bind the amino-terminal RRMs (amino

acids 1-197). Thus, substrate specificity depends on the presence of both RRMs (17,99), and

the RS domain is not a major determinant of binding specificity (83).

SF2/ASF and EIAV Rev

As with other retroviruses, EIAV utilizes the splicing machinery to produce the viral

RNAs necessary for replication. EIAV is also a lentivirus and produces the regulatory

protein Rev, which is necessary for the cytoplasmic expression of the unspliced and singly

spliced viral mRNAs. Interestingly, exon 3 of the four-exon, bicistronic viral mRNA

(mRNAl, Figure 4) contains a purine-rich sequence that functions as both an ESE and as an

EIAV RRE (ESE/RRE) (5,34). This ESE/RRE was sufficient for SR protein binding (18),

and was necessary for exon inclusion, Rev binding, and Rev-dependent nuclear export (5).

The SR protein that specifically binds the ESE/RRE is SF2/ASF (18,34), and the ESE/RRE

contains multiple sequences that conform to the SF2/ASF consensus RNA binding motif

RG A AG A AC.

A mutational analysis of the ESE/RRE has shown that the sequences that bind Rev

also strongly assist in ESE-mediated splicing, and mutations that inhibit ESE function also

inhibit RRE function (3). However, mutation of the ESE/RRE does not exclusively knock

out the function of both the ESE and RRE. Thus, it is thought that the RNA binding sites for

SF2/ASF and Rev on the ESE/RRE overlap, but are not the same. It is clear that Rev can

inhibit exon 3 inclusion in the bicistronic viral mRNA (5,62), and SF2/ASF can inhibit Rev-

mediated nuclear export activity (5). These results suggest that Rev and SF2/ASF

competitively bind the ESE/RRE, which regulates splicing and viral mRNA expression.

Because Rev is absolutely required for virus replication, SF2/ASF may also inhibit EIAV

replication. Therefore, the interaction of Rev and SF2/ASF with the ESE/RRE may

modulate EIAV replication, and ultimately contribute to viral persistence and pathogenesis.

19

Overall Goal

The lentiviral protein Rev is absolutely required for EIAV replication, and factors that

inhibit Rev function would be expected to inhibit virus replication. The cellular SR protein

SF2/ASF inhibits EIAV Rev function (5). Rev and SF2/ASF are regulators of viral mRNA

splicing and expression, and they both play a role in EIAV alternative splicing. The goal of

this research is to characterize the interactions between EIAV and cellular SR proteins that

modulate virus replication. It is our hypothesis that SF2/ASF inhibits Rev nuclear export

activity and virus replication. To test this hypothesis, we have undertaken the following

specific aims:

Specific Aims:

1. Determine the effect of Rev-mediated alternative splicing on EIAV Tat activity.

(Chapter 2)

2. Determine the effect of SF2/ASF expression on Rev-dependent nuclear export.

(Chapter 3)

3. Determine the effect of SF2/ASF expression on EIAV replication.

(Chapter 3)

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34

CHAPTER 2. FUNCTIONAL CHARACTERIZATION OF TAT ACTIVITY FROM

EIAV ALTERNATIVELY SPLICED MESSENGER RNAS

A paper to be submitted to the journal Virus Genes

Gregory Park, Michael Belshan, Susan Schommer, and Susan Carpenter

Abstract

Similar to other Antiviruses, equine infectious anemia virus (EIAV) encodes the

regulatory proteins Tat and Rev, which regulate virus gene expression. Tat up-regulates

transcription and Rev binds at a region of viral RNA called the Rev responsive element

(RRE), and exports incompletely spliced viral transcripts into the cytoplasm. Tat and Rev

are translated from a four-exon bicistronic mRNA, which is the predominate mRNA

expressed early after infection. Tat translation is initiated at a CUG in exon 1, and only

through a leaky scanning mechanism is Rev translation initiated at the first AUG located in

exon 3. Exon 3 also contains the EIAV RRE. Rev mediates the expression of a three-exon,

alternatively spliced mRNA that lacks exon 3 and encodes only Tat. To determine the effect

of Rev-mediated alternative splicing on Tat expression, we used transient assays to

functionally analyze alternative spliced mRNAs. There was no significant difference in Tat

activity between monocistronic and bicistronic cDNAs, suggesting that Tat expression is not

different between the three-exon and four-exon EIAV mRNAs. Thus, the presence or

absence of Exon 3 did not affect Tat activity, suggesting that neither the RRE in rev exon 1

nor translation initiation of Rev affect Tat expression.

35

Introduction

Retroviruses are single stranded, positive sense RNA viruses that use reverse

transcriptase to create dsDNA from their ssRNA genome. The single viral promoter of the

integrated proviral dsDNA produces unspliced viral pre-mRNA. Retroviruses utilize both

viral and host cell factors to express unspliced and incompletely spliced viral mRNAs.

Members of the lenti virus subfamily of retroviruses encode two proteins, Tat and Rev, which

regulate viral gene expression (reviewed in 11,27,32). Rev acts post-transcriptionally to

facilitate the nuclear export of unspliced and incompletely spliced mRNAs, and is absolutely

required for virus replication (reviewed in 11,17,27). Rev functions by entering the nucleus,

binding viral mRNAs at a region of RNA called the Rev responsive element (RRE), and

exporting the viral mRNAs into the cytoplasm (7,13,14,26,36,37). Tat functions by entering

the nucleus and, in association with cyclin T1 and cyclin-dependent kinase 9 (Cdk9) (3,34),

binds to a bulged, stem-loop structure found at the 5' terminus of all viral mRNAs called the

Tat activation region (TAR) (8,12,16,28,29). Cdk9 phosphorylates the C-terminal domain of

RNA polymerase II (RNAPII) to shift transcription from initiation to elongation (22,35,38).

Therefore, Tat promotes RNAPII elongation, and can enhance transcription of viral mRNA

50-100 fold.

Equine infectious anemia virus is a member of the lenti virus subfamily of retroviruses

that produces Tat and Rev from a four-exon, bicistronic mRNA (Figure 1) (24,31). Tat is

translated from exon 1 and exon 2, and Rev is translated from exon 3 and exon 4

(9,10,24,25,31). Interestingly, Tat translation is initiated at a CUG start codon in exon 1

(4,9,25,30), whereas Rev translation initiates at an AUG start codon in exon 3 through a

leaky ribosomal scanning mechanism (4). In the absence of Rev, the four-exon mRNA is the

only cytoplasmically expressed mRNA (mRNAl, Figure 1) (23,30). The presence of Rev

results in the expression of the other EIAV mRNAs (mRNA3, mRNA4, mRNA5) including a

monocistronic, three-exon mRNA, encoding only Tat (mRNA 2) (23). The monocistronic

mRNA is identical to the bicistronic mRNA, except it lacks exon 3.

Leaky ribosomal scanning proposes that only some ribosomes stop to initiate

translation at non-AUG start codons or AUG start codons in a weak context (A or G not at

position -3 and G not at +4), while most continue scanning downstream (20). There are

factors that affect leaky scanning translation other than the context of the start codon. RNA

secondary structures can affect translation initiation in bicistronic messages, depending on

their location. RNA secondary structures located downstream of the first initiation site can

enhance translation of the first cistron by slowing ribosomes enough for codon/anti-codon

base pairing to occur (19).

Exon 3 of the four-exon bicistronic mRNA contains not only the translation initiation

site for Rev, but also contains the EIAV RRE, which is suggested to have secondary structure

(6,15). Secondary structure would be consistent with other RREs, such as HIV's RRE, which

is a large secondary structure (5,18,21). Thus, the three-exon alternatively spliced RNA may

differ from the four-exon mRNA in Tat expression due to the absence of exon 3, which may

contain RNA factors that affect translation of the upstream cistron. To determine the effects

of EIAV Rev-mediated alternative splicing on Tat expression, transient expression assays

were used to compare the functional activity of Tat among alternatively spliced EIAV

cDNAs. There was no significant difference in Tat activity in cells transfected with the

monocistronic or the bicistronic cDNAs, suggesting that Tat expression is not different

between the monocistronic and bicistronic EIAV mRNAs that encode Tat. Therefore, the

37

results suggest that neither the RRE nor translation initiation at the Rev AUG affect Tat

translation.

Materials and Methods

Plasmids. EIAV MA-1 cDNAs (4x+, 3x+, 4x-) were amplified by RT-PCR of total

RNA isolated from Cf2Th cells transfected with Rev+ or Rev- proviral DNA using the 5'

primer, CGCAGACCCTACCTGTTG and the 3* primer, TAGCCTGCTATGCGTCCTAC

(Figure 2). The cDNA products were TA-cloned into pCR3.1 (Invitrogen, Carlsbad, Calif.)

and confirmed by sequence analysis (DNA Sequencing and Synthesis Facility, Iowa State

University). Transcription of the cDNAs in pCR3.1 is under control of the CMV promoter.

ETat-M was constructed by David Derse in pRSPA-S (pRSETAT-M) (9). Exon 1 of the Tat

cDNA in pRSETAT-M is missing the first 38 nucleotides and starts at an engineered AUG

initiation site. Transcription of pRSETAT-M is under control of the RSV promoter. The

plasmid pCHl 10 (Amersham Pharmacia, Buckinghamshire, UK) produces (3-galactosidase.

The LTR-CAT reporter plasmid pCATLTREIAV-1 contains the EIAV LTR upstream of a

chloramphenicol acetyltransferase (CAT) gene. The plasmid pcDNA3 (Invitrogen) was used

as a negative control and produces no Tat.

Transient transaction assays. Transient transfections were performed in canine

fetal thymus (Cf2Th) cells, which support both EIAV Tat activity and EIAV replication (33).

Cf2Th cells were maintained in Dulbecco's modified Eagle media supplemented with 10%

fetal calf serum and penicillin/streptomycin. All transfections were performed with the

transfection reagent LipofectAMINE (Life Technologies/Invitrogen, Carlsbad, CA)

according to reagent protocols. Briefly, 0.2 \xg of individual cDNA plasmids were co-

transfected into cells with 0.2 |xg pCHl 10,1.0 jig of pCATLTREIAV-1, and an amount of

pUC19 to equalize the amount of DNA used in all transfections. Two days post-transfection,

cells were harvested, lysed by freeze/thaw, and clarified lysates were assayed for transfection

efficiency by their (3-galactosidase activity. Normalized amounts of cell lysate were then

assayed for CAT expression with a commercially available CAT enzyme-linked

immunosorbent assay (ELIS A) kit (Roche Molecular Biochemicals, Indianapolis, IN). Pilot

assays quantified CAT from pRSETAT-M transfected cells, and results were used to

determine the parameters to assay all the transfected cell lysates. Initial assays determined a

range of Tat activity from co-transfection of increasing amounts of pRSETAT-M or p3x+

with pCATLTREIAV -1 (data not shown). Results were used to determine the working

amount of Tat cDNA plasmid.

Results and Discussion

EIAV Tat translation is initiated at a non-standard CUG in exon 1 of both the three-

exon and four-exon EIAV mRNAs. Rev translation initiates in exon 3 at the first AUG of the

EIAV four-exon, bicistronic viral mRNA. Thus, leaky scanning of the CUG initiation codon

permits translation of Rev (4). Interestingly, Rev mediates the expression of the three-exon

mRNAs that lack exon 3 and encode only Tat. Exon 3 contains a purine-rich sequence that

functions as both an ESE and an RRE. Typical lenti viral RRE's are highly structured RNA

elements. It is possible that initiation at the Tat CUG is altered depending on the presence or

absence of the AUG initiation site and/or RNA secondary structure in exon 3. If so, Rev-

mediated alternative splicing may be a novel mechanism to increase Tat activity. To

determine if EIAV alternative splicing results in mRNAs that differ in Tat activity, cDNAs

were constructed and tested for Tat activity in an in vitro transient expression assay. EIAV

Tat constructs, 3x+ and 4x+ (Figure 2), represent the three-exon and four-exon mRNAs,

respectively, and differ in the absence and presence of exon 3. Thus, 3x+ encodes only Tat,

and 4x+ encodes both Tat and Rev. We also constructed a cDNA that contain exon 3, but

produce no Rev due to a stop codon in exon 4. This would allow us to differentiate any

effect due to Rev or exon 3. Thus, 4x- encodes a truncated Rev protein (amino acids 1-79)

that contains only the identified nuclear export signal. The truncated Rev protein lacks

amino acids 80-165, which includes the domains required for RNA binding and nuclear

localization, and the protein is expected to be non-functional. The ETat-M construct has an

engineered AUG start codon for Tat instead of the wild-type CUG, and was used as a

positive control for Tat activity.

Less than 2-fold differences in levels of CAT were observed among the cDNA

constructs. There was no significant difference in Tat activity between the 3x+ and 4x+

constructs (P>0.05) (Figure 3), which indicates that Tat activity is the same between the

monocistronic and bicistronic EIAV Tat mRNAs. Co-transfection of the CAT reporter with

the 4x- construct resulted in levels of CAT not significantly different from 3x+ (P>0.05).

Together, these data suggest that the presence of exon 3 has little to no effect on Tat activity

(Figure 3). Interestingly, the 4x- construct had significantly higher Tat activity than the 4x+

construct (P<0.05). While this suggests that Rev inhibited Tat activity, there is no significant

difference in Tat activity between the 3x+ and 4x+ constructs. In addition, trans-

complementation experiments showed that Rev does not significantly affect Tat activity (data

not shown). It is possible that the presence of a truncated Rev protein may affect Tat

activity. Together, the data indicate that there is no difference in Tat activity between the

40

monocistronic and bicistronic EIAV mRNAs, and suggest that exon 3 does not affect Tat

expression. Thus, EIAV Rev-mediated alternative splicing may be a mechanism to express

mRNAs that produce Tat without producing Rev, which may play a role in regulating both

viral protein and mRNA expression.

Rev mediates exclusion of exon 3, and the subsequent expression of the alternatively

spliced, three-exon mRNAs that encode only Tat. Recently, an exon splicing enhancer has

been mapped to exon 3 (2,6,15). The ESE sequence also functions as a RRE (ESE/RRE) and

the sequences of the ESE/RRE bind Rev. The cellular splicing protein SF2/ASF also binds

the ESE/RRE and assists in exon 3 inclusion (2,15). The mechanism that results in

expression of the alternatively spliced, three-exon mRNA is not clear, but current models

suggest that exon 3 exclusion is the result of the inhibition of splicing, due to the binding of

Rev at the ESE/RRE. The biological significance of the three-exon mRNA in virus

replication is not known, but our data indicate that exon 3 had no effect on Tat activity. This

does not necessarily mean that production of a second mRNA species encoding Tat plays no

role in EIAV replication. Indeed, Tat is produced at very low levels, yet has profound affects

on viral transcription (3). Our data suggests that Rev-mediated alternative splicing is not a

mechanism to increase Tat expression. Thus, further investigations are necessary to better

understand if the generation of the alternatively spliced mRNA is important in EIAV

replication.

41

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34. Wei, P., M.E. Garber, S. Fang, W.H. Fischer, and K.A. Jones. 1998. A novel

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48

Figure Legends

Figure 1. The five major mRNAs of EIAV and their encoded proteins. Messenger RNA1 and

mRNA2 encode the regulatory proteins Tat and Rev. Messenger RNA3 encodes a truncated

transmembrane protein (Ttm) (1). Rev facilitates the nuclear export of incompletely spliced

mRNA4 and unspliced mRNA5, which encode the structural proteins of the virus. The

unspliced mRNA5 also serves as the genome for new virions. The boxes of the genome

indicate the open reading frames. The thick, horizontal lines of the mRNAs indicate the exon

sequences of the mRNAs, whereas the diagonal lines indicate the intron sequences that are

spliced out. The proteins the mRNAs encode are listed.

Figures 2. EIAV genome and cDNA constructs. (A) EIAV genome and four-exon mRNA

that encodes both Tat and Rev. Exons 1 and 2 encode Tat and exons 3 and 4 encode Rev.

The boxes of the genome indicate the open reading frames, whereas the boxes of the RNA

indicate the exons. (B) Expression plasmids used to evaluate the effect of alternative splicing

on Tat activity. Promoters and protein products produced from the constructs created are

shown. The 4x+ construct produces both Tat and Rev. The 3x+ construct is the same as the

4x+ construct except that it lacks exon 3, and produces only Tat. The diagonal lines of 3x+

indicate exon 3 exclusion in the cDNA. The 4x- construct contains a stop codon in exon 4

and encodes a truncated Rev protein that contains only the nuclear export signal. The ETat-

M construct produces Tat from an engineered AUG start codon instead of the wild-type

CUG, and transcription is under control of the RSV promoter. The boxes of the genome

indicate the open reading frames, whereas the boxes of the constructs indicate the exons

included.

49

Figure 3. Tat activity of alternatively spliced mRNAs. (A) Linear range of Tat activity from

transfected pRSETAT-M and p3x+. (B) EIAV cDNA constructs produce Tat that

transactivates the LTR-CAT reporter to produce CAT. The plasmid pcDNA3 produces no

protein, and the results indicate the background ground level of LTR-CAT reporter

expression. The results represent the mean of at least nine independent transfections per

construct, and the error bars represent the standard error of the mean. The asterisks indicate a

significant difference, as determined by ANOVA, between the results from cells transfected

with either p4x+ or p4x- (P<0.05).

50

0 poI

mRNA

gag gp9Q | gp45~\

Protein

Tat, Rev

Tat

Ttm

gp9Q gp45 Env

gag Gag, Pol

Figure 1.

51

Expression Piasmids

4x+

3x+

4x-

ETat-M

Promoter Exons CUG AUG

CMV -[ggg;

CMV H3H C CMV

RSV

yUAG

h|2|3| | „

HHB

Proteins

Tat, Rev

Tat

Tat

Tat

Figure 2.

52

pcDNAS |

ETat-M 4"

3x+ +

4x+ 4 ' *

4x- -h * i i i i •

0 20 40 60 80 100

pg CAT

Figure 3.

53

CHAPTER 3. SF2/ASF INHIBITS EQUINE INFECTIOUS ANEMIA VIRUS

REV ACTIVITY AND VIRAL REPLICATION

A paper to be submitted to the journal Molecular and Cellular Biology

Gregory Park and Susan Carpenter

Abstract

Complex retroviruses encode the essential regulatory protein Rev/Rex, which

functions by entering the nucleus, binding viral mRNAs at the Rev/Rex responsive element

(RRE), and exporting the singly spliced and unspliced viral mRNAs necessary for virus

replication into the cytoplasm. Equine infectious anemia virus (EIAV) encodes Rev in exons

3 and 4 of a four exon, bicistronic viral mRNA. A purine-rich sequence in exon 3 functions

as an exon splicing enhancer (ESE), and binds the cellular SR protein SF2/ASF. The ESE

also binds Rev and functions as the EIAV RRE (ESE/RRE). Here, we show that SF2/ASF

inhibits Rev-dependent nuclear export activity and EIAV replication. Inhibition mapped to

the RNA binding domain of SF2/ASF and correlated with the RNA binding specificity.

These results indicate that SF2/ASF binding viral mRNAs inhibits Rev-dependent nuclear

export activity and EIAV replication, and suggest that the SF2/ASF and Rev compete for

binding the ESE/RRE.

54

Introduction

Retroviruses have a limited genome size and employ a variety of mechanisms to

express unspliced or differentially spliced viral mRNAs transcribed from a single promoter.

One such mechanism involves the use of non-consensus splice sites and czj-regulatory

sequences to produce unspliced and singly spliced viral mRNAs by inefficient splicing.

Other mechanisms utilize viral and cellular proteins that act in trans to regulate the

cytoplasmic expression of incompletely spliced viral mRNAs. Members of the lentivirus

subfamily of retroviruses encode the protein Rev to assist in the cytoplasmic expression of

the singly and unspliced viral mRNAs. The prototypical Rev is human immunodeficiency

virus type 1 (HIV-1) Rev, which functions by entering the nucleus, binding singly and

unspliced viral mRNAs at a sequence in the env called the Rev responsive element (RRE)

(14,55), multimerizing (37,54), and exporting the mRNAs into the cytoplasm through the

exportin 1 (Crml) nuclear export pathway (16,18). The RRE-containing viral mRNAs

encode the structural proteins necessary for replication, and the unspliced mRNAs also serve

as the genome for new virions. The cytoplasmic expression of these viral RNAs is dependent

on Rev and, therefore, Rev is absolutely required for virus replication.

Equine infectious anemia virus (EIAV) is a lenti virus similar to HIV, and also

encodes a Rev that it is necessary in replication. Early in infection, EIAV expresses a

multiply spliced, four-exon, bicistronic mRNA (mRNAl, Figure 1). The first and second

exons encode the transcriptional activator protein Tat, and the third and fourth exons encode

Rev (46). EIAV Rev (ERev) is a 165 amino acid protein with little amino acid homology to

other lentiviral Rev proteins. It is functionally homologous* to HIV-1 Rev, yet not as well

characterized (19,32). As with other Rev's, ERev enters the nucleus, binds RRE-containing

mRNAs, and is necessary for the nuclear export of the singly and unspliced transcripts

(mRNAs 3&4, Figure 1) through an exportin 1-mediated pathway (16-18,45). In addition,

the presence of ERev results in the expression of an alternatively spliced viral mRNA that

lacks exon 3. The rev AUG start codon is located in exon three and the alternatively spliced

mRNA2 (Figure 1) encodes only Tat.

Exon 3 contains a purine-rich sequence that functions as both an exon splicing

enhancer (ESE) and as a RRE (ESE/RRE). Exon splicing enhancers are c à-acting,

intraexonic sequences that assist in exon recognition and inclusion during splicing by

interacting with a family of cellular proteins, called the SR proteins (reviewed in 5,20,33).

Members of the SR protein family share a modular, functional domain structure of one or

two amino-terminal RNA recognition motifs (RRMs) and a carboxy-terminal RS domain that

consists of a number of arginine-serine dipeptide repeats (12). SR proteins function by

binding ESEs and assisting components of the splicing machinery to recognize non-

consensus splice sites through protein-protein interactions (reviewed in 5,10,49). SR protein

activity is both substrate and concentration-dependent, and their intracellular concentration

has been shown to vary in a number or tissues and cellular activation states (22,25,53).

Many SR proteins have been identified in a variety of both plants and animals. One SR

protein, SF2/ASF, is highly conserved in mammals and has numerous homologs in other

diverse species including birds, plants, worms, and insects (1,2,8,29,30,53). SF2/ASF binds

the ESE/RRE in EIAV rev exon 1, which suggests it is the SR protein partner to the ESE

(13,21).

In vitro splicing assays demonstrated that the purine-rich ESE motif was required for

exon three splicing, and that splicing was inhibited by GST-ERev fusion protein (4). The

current model for alternative splicing of mRNA2 is that binding of ERev to the ESE/RRE

inhibits SF2/ASF-dependent exon 3 inclusion (4,13). Interestingly, our previous results

suggested that SF2/ASF can inhibit Rev-dependent nuclear export (4). Here, we further

investigate the effects of SF2/ASF on EIAV Rev-dependent nuclear export. SF2/ASF did not

affect the expression of Rev, but inhibited Rev-dependent nuclear export activity and EIAV

replication in a dose-dependent manner. Further, the inhibition mapped to the RNA binding

domain and correlated with RNA substrate specificity. These data indicate that SF2/ASF

binding the viral RNA inhibits both Rev activity and EIAV replication, and suggest that Rev

and SF2/ASF are functionally competitive. If so, the intracellular concentrations of SR

proteins may be an important factor in the regulation of EIAV replication.

Materials and Methods

PGR and Plasmid Construction. All plasmids and constructs were confirmed by

sequence analysis (Iowa State University DNA Synthesis and Sequencing Facility). PCRs

for the construction of plasmids were carried out using 1 jaM primers and standard protocols

as per the manufacturer (Perkin Elmer, Foster City, CA). Unless otherwise indicated, PCRs

consisted of 30 cycles of 2 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1

min of extension at 72°C, followed by an additional cycle with a 7 min extension.

The expression plasmid pERev contains a Rev cDNA derived from a MA-1 isolate of

EIAV previously described (9). pAERev was constructed by the digestion and ligation of

two Rev fragments to produce a cDNA that encodes ERev without amino acids 19-23. The

upstream fragment, which encodes amino acids 1-18, was PCR amplified using the 5' primer

A AT ACG ACTC ACT ATAG and the 3' primer GAGTCTAGATTCTTCTTTCAG. The

57

downstream fragment, which encodes amino acids 24-165, was PGR amplified using the 5'

primer GAGTCTAGAGACTGGTGGAAA and the 3' primer

GAGGAATTCTCATAAATGTTTCCTCCT. The underlined nucleotides indicate an

introduced Xbal site at the end and beginning of the two respective fragments. The resulting

PCR products were digested with Xbal, ligated together using standard protocols, and

amplified with the 5' primer TAAGAACAGCATGGCAGAATCGAAG and the 3' primer

GAGGAATTCTCATAAATGTTTCCTCCT. The resulting cDNA was TA cloned into the

eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, CA).

A recombinant baculovirus that expresses SF2/ASF was generously provided by Tom

Maniatis (Harvard University). The SF2/ASF cDNA was amplified from purified

baculovirus DNA using the 5' primer ACCGCCATGTCGGGAGGT and the 3' primer

ATCTTATGTACGAGAGCG, and TA cloned into pCR3.1. The SF2/ASF deletion mutants

pSF2ARS and pSF2ARRMS were also constructed by PCR amplification from recombinant

baculovirus DNA and TA cloning into pCR3.1. The SF2ARS cDNA was constructed by

amplifying a SF2/ASF fragment that encodes amino acids 1-195 using the 5' primer

ACCGCCATGTCGGGAGGT and the 3' primer GGGCTAATCAACTTTAACCCGG. The

underlined codon of the 3' primer indicates the anti-sense introduced stop codon after amino

acid 195. The SF2ARRMS cDNA was constructed by amplifying a SF2/ASF fragment that

encodes amino acids 196-248 using the 5' primer

ACCGCC ATGCCC AG A AGTCC AAGTT AT and the 3' primer

ATCTTATGTACGAGAGCG. The underlined codon in the 5' primer indicates a start codon

introduced before amino acid 196.

58

Both pSF2ARRMl and pSF2ARRM2 were constructed by PCR amplification from

pSF2/ASF and TA cloning into pCR3.1. The SF2ARRM1 cDNA was constructed by

amplifying a SF2/ASF fragment that encodes amino acids 107-248 using the 5' primer

ACCGCCATGGCTCCCCGAGGTCGC and the 3' primer

GGATCCATCTTATGTACGAGAGCG. The SF2/ASF deletion mutant SF2ARRM2 was

constructed by amplifying the RRM1 and RS domain cDNAs with primers that introduce a

Pvul restriction site into both. The RRM1 cDNA encoding amino acids 1-97 was amplified

with the 5' primer ACCGCCATGTCGGGAGGT and 3' primer

ATCGATCGTCGGCCTGTTCC. The RS domain cDNA encoding amino acids 198-248

was amplified with the 5' primer TACGATCGCCAAGTTATGGA and 3' primer

GGATCCATCTTATGTACGAGAGCG. The underlined nucleotides in the primers indicate

the Pvul restriction site. The PCR products were digested with Pvul and ligated together

using standard protocols. The full length SF2ARRM2 cDNA was then amplified using the 5'

primer ACCGCCATGTCGGGAGGT and the 3' primer

GGATCCATCTTATGTACGAGAGCG, and the resulting cDNA was cloned into pCR3.1.

Subcellular localization of SF2/ASF and the SF2/ASF deletion mutants were assessed

using GFP fusion proteins. Restriction digestion of the flanking EcoRl and BamHl sites in

the multiple cloning site of pCR3.1 produced cDNA fragments that were cloned into the

multiple cloning site of an EcoRl and BamHl digested GFP vector, pEGFP-C2 (BD

Biosciences Clontech, Palo Alto, CA). One (Xg of each GFP fusion construct was transfected

into Cf2Th cells, and 48 hours post-transfection, nuclear localization was observed by light

microscopy using an inverted Nikon Diaphot fluorescence microscope with a 40X objective,

a 100W high-pressure mercury lamp, and epifluorescence filters.

59

A plasmid containing the cDNA of SC35 was obtained from ATCC (ATCC#95691),

and the cDNA was amplified and TA cloned into pCR3.1 using the 5' primer

GATTTAGGTGACACTATAG and the 3' primer TAATACGACTCACTATAGGG.

Transfections. Transfections were performed according to the reagent protocol with

TransIT-LTl transfection reagent (Minis, Madison, WI) and canine fetal thymus (Cf2Th)

cells. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)

supplemented with 10% fetal calf serum and penicillin-streptomycin. In all transfections,

pcDNA3 (Invitrogen, Carlsbad, CA) was used to equalize the amount of DNA per

transfection, and TransIT-LTl was used at a ratio of 2 jLtl:l|Xg DNA.

Western blot analyses. Cf2Th cells were transfected with 100 ng of pAERev, 200

ng of the pCHllO, increasing concentrations (50,100, or 500 ng) of pSF2/ASF, and a

varying concentration of pcDNA3. Two days post-transfection, cells were harvested,

resuspended in a 100 fxl of 0.25 M Tris-HCl (pH 7.5), and 50 (xl was lysed by freeze/thawing

and assayed for (3-galactosidase activity. The remaining volume was lysed by adding 2X

lysis buffer (63 mM Tris-HCl, pH 6.8,1% (w/v) SDS, 10% (v/v) |3-mercaptoethanol, 0.004%

(w/v) bromophenol blue). Samples were normalized for (3-galactosidase activity, and

electrophoresed in a 12% SDS-PAGE gel. Duplicate gels were blotted, blocked with a 1%

gelatin solution, and reacted with either EIAV convalescent horse serum or a monoclonal

anti-SF2/ASF antibody (Zymed Laboratories, San Francisco, CA). Blots were then reacted

with 125I-protein G (Amersham Biosciences, Allington Heights, IL) or 125I-anti-mouse Ig

(Amersham Biosciences, Allington Heights, IL) and analyzed with a Personal Molecular

Imager FX Phosphorlmager (Biorad, Hercules, CA) using Quantity One (Biorad, Hercules,

CA) imaging and analysis software. For Western blot analyses of SC35, a commercially

60

available anti-SC35 monoclonal antibody (Sigma, St. Louis, MO) was used. One fig of

pSC35 was transfected into Cf2Th cells, and two days post-transfection Western blot

analyses of transfected cell lysates showed protein expression of SC35 higher than

background levels of non-transfected cell lysates.

CAT assays. The Rev nuclear export reporter pERRE-1 A has been previously

described (4). Briefly, pERRE-1 A contains EIAV nucleotides 5281 to 5795 downstream of a

CAT gene. Thus, pERRE-1 A includes the identified EIAV RRE (nucleotides 5485 to 5540),

(Figure IB). The CAT gene and RRE sequences are flanked by an upstream splice donor

and a downstream splice acceptor. In the presence of Rev, reporter transcripts are bound by

Rev, exported into the cytoplasm, and CAT is expressed. In the absence of Rev, reporter

transcripts are spliced, removing the CAT gene and RRE. Thus, no CAT is expressed.

Cf2Th cells were transfected with 1 jxg of pERRE-1 A, 0.2 pig of pCHl 10 and the required

expression plasmids. Two days post-transfection cells were harvested and resuspended in 0.5

ml of 0.25 M Tris-HCl (pH 7.5). Cells were lysed by freeze/thawing, and lysates were

assayed for p-galactosidase activity to normalize for transfection efficiency. Normalized

lysates were assayed for CAT expression with a commercially available CAT enzyme-linked

immunosorbent assay (ELIS A) kit (Roche Molecular Biochemicals, Indianapolis, IN). Pilot

assays were performed with pAERev and pERRE-1 A co-transfected cell lysates to determine

assay conditions within the linear range of the CAT ELIS A. The results of pilot assays were

used to adjust the normalized amounts of cell lysates to assay conditions, and all sample

lysates were subsequently assayed for CAT.

Virus replication assays. The EIAV infectious molecular clone pSPEIAV-19R was

kindly provided by Susan Payne (University of Texas, Arlington) and has been previously

described (38). Cf2Th cells were co-transfected with 2 fig of pSPEIAV-19R, 0.2 jig of

pCHl 10, and the various expression plasmids. Supernatant was collected and cells were

harvested, lysed, and assayed for P-galactosidase activity. Cell-free, infectious virus was

titered on equine dermal (ED) cells with a focal immunoassay using a chromogenic method

of detection as previously described (44). Briefly, harvested culture supernatant samples

were clarified by centrifugation at 2000 x g for 5 min, serially diluted in DMEM containing

polybrene (8 jig/ml), and added to cultures of ED cells. Five days post-inoculation, ED cells

were washed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4), and

fixed with 100% methanol. Cells were incubated with an EIAV convalescent horse serum,

followed by incubation with a horseradish peroxidase-conj ugated goat anti-horse IgG

polyclonal antibody, and incubation with the AEC substrate (0.2 mg/ml 3-amino-9-

ethylcarbazole, 0.01% H202, 0.05 M NaOAc, pH 5.0). Focus forming units (FFUs) were

counted and results were normalized to P-galactosidase activity.

Reverse transcriptase (RT) assays were performed as previously described (9).

Briefly, 10 |ll of clarified culture supernatant was mixed with 30 (il RT cocktail (50 mM

Tris-HCl, pH 7.9,75 mM KC1, 2 mM dithiothreitol, 5 mM MgClz, 0.05% Nonidet P-40,4

|ig/ml oligo(dT), 5 jug/ml polyriboadenylic acid, 20 jiCi/ml [a-32P]dTTP), incubated at 37°C

for 2 hours, and 8 fil was spotted onto DE81 paper. After washing, the DE81 paper was

analyzed using a phosphorlmager and software as described above. Results were normalized

to P-galactosidase activity.

62

Results

SF2/ASF inhibits EIAV Rev-dependent nuclear export. In previous studies, we

determined that increasing concentrations of the SR protein SF2/ASF resulted in a dose-

dependent decrease in Rev nuclear export activity (4). The first exon of the Rev cDNA

contains the EIAV RRE which binds SF2/ASF (13) and functions as an ESE (ESE/RRE) (4).

Therefore, it is possible that SF2/ASF binding to rev mRNA could affect Rev expression in

transient assays. The transient assays are done in the linear range of Rev activity, in which

the levels of Rev protein are below the limits of our detection. To rule out the possibility that

the observed decrease in activity was due to SF2/ASF inhibition of Rev protein expression

rather than an inhibition of Rev function, we constructed a Rev cDNA expression plasmid in

which we deleted the poly-purine stretch in the ESE/RRE, thus eliminating the SF2/ASF

binding site (AERev). Cf2Th cells were co-transfected with pAERev in the presence of

increasing amounts of pSF2/ASF, and cell lysates were analyzed by Western blot analysis.

Increasing concentrations of pSF2/ASF did not decrease levels of AERev protein (Figure

2A). The nuclear export activity of AERev was comparable to wild-type Rev (Figure 2B),

although it was less efficient in the lower range of concentrations tested. Therefore, AERev

was used to further examine the effect of SF2/ASF on Rev function.

Increasing concentrations of pSF2/ASF resulted in a significant, dose-dependent

decrease in Rev nuclear export activity (P<0.05) (Figure 3A). In the absence of AERev,

there was little change in the expression of CAT over the range of pSF2/ASF concentrations

tested. Therefore, the inhibition of nuclear export by SF2/ASF was not due to an

independent effect on the CAT reporter plasmid, but to a specific inhibition of Rev function.

CAT levels were restored when increasing amounts of pAERev were co-transfected with an

inhibitory amount of pSF2/ASF (Figure 3B). This suggested that AERev and SF2/ASF

compete for a factor in the Rev export pathway.

The amino-terminal RNA recognition motifs of SF2/ASF are necessary and

sufficient to inhibit Rev-dependent nuclear export. SR proteins are comprised of

modular, functional domains, including one or two amino-terminal RRMs and a carboxy-

terminal RS domain (12). The RRMs function typically in RNA binding, whereas the RS

domain mediates protein-protein interactions. SELEX analyses of individual members of the

SR protein family have shown differences in RNA binding specificity, depending on the

identity and number of RRM domains (Figure 4). For example, the SR protein SF2/ASF

contains two RRMs, and the RNA binding specificity differs depending on whether one or

both RRMs are present (48). The ESE/RRE in EIAV rev exon 1 contains multiple copies of

a near perfect binding site for SF2/ASF. To determine if binding to the ESE/RRE plays a

role in the inhibition of Rev dependent nuclear export activity, we constructed a series of

SF2/ASF deletion mutants that lack either the RS domain or one or both of the RRMs (Figure

5A). Nuclear localization of each construct was confirmed using GFP-fusion proteins (data

not shown).

Each SF2/ASF mutant was tested for effect on Rev nuclear export activity in transient

expression assays (Figure 5B). In the absence of AERev, none of the constructs had an

inhibitory effect on CAT expression from the reporter plasmid (data not shown). Deletion of

the RS domain (SF2ARS) resulted in a significant inhibition of Rev activity to levels

equivalent to that of wild-type SF2/ASF (Figure 5B). However, deletion of one or both of

the amino-terminal RRMs (SF2ARRMS, SF2ARRM1, SF2ARRM2) had no significant effect

on Rev activity (P>0.05). To rule out that these results reflected differences in levels of

protein expression, assays were done using increasing concentrations of deletion plasmids.

Co-transfection of up to 10 fold more of either pSF2ARRMlor pSF2ARRM2 resulted in no

significant inhibition of Rev activity (data not shown). Therefore, the presence of

RRM1/RRM2 was both necessary and sufficient for SF2/ASF inhibition of Rev activity. The

RNA binding specificity of both SF2/ASF and SF2ARS is conferred by RRM1/RRM2, and

distinct from the RNA consensus binding specificity conferred by RRM1 alone (Figure 4).

Moreover, the consensus RRM1/RRM2 binding site is nearly identical to multiple parts of

the EIAV ESE/RRE. The correlation between RNA binding specificity and Rev inhibition

suggests that binding to the viral RNAs at the ESE/RRE is required for SF2/ASF inhibition

of Rev activity.

The amino-terminal RNA recognition motifs of SF2/ASF are necessary and

sufficient to inhibit EIAV replication. Rev is absolutely required for virus replication, and

factors that inhibit Rev would be predicted to inhibit EIAV replication. To determine if

SF2/ASF could inhibit production of infectious virus, Cf2Th cells were transfected with an

infectious molecular clone in the presence or absence of pSF2/ASF. In cells transfected with

the proviral clone and pcDNA3, the supernatant reverse transcriptase (RT) activity increased

over 48 hours. In the presence of pSF2/ASF, RT activity was only slightly higher than

background levels over the same time period (Figure 6A), such that by 48 hours, there was

15-fold less RT activity in pSF2/ASF-transfected cells as compared to cells transfected with

pcDNA3. When increasing amounts of pSF2/ASF were co-transfected, we observed a dose-

dependent decrease in virus titer, with nearly a 2-log reduction in virus titer in the presence

of 1 jig of transfected pSF2/ASF (Figure 6B). To map the domains of SF2/ASF that inhibit

EIAV replication, each SF2/ASF deletion construct was co-transfected with the proviral

clone. SF2ARS inhibited virus replication similar to wild-type SF2/ASF, whereas no

significant inhibition was observed with SF2ARRMS, SF2ARRM1, or SF2ARRM2 (P>0.05)

(Figure 6C). Therefore, the presence of RRM1/RRM2 was necessary and sufficient to inhibit

virus replication, indicating that the inhibition of EIAV replication correlated with SR protein

RNA binding specificity.

Results using SF2/ASF deletion constructs indicated that inhibition of EIAV Rev

activity correlated with RNA binding specificity of the SR protein. SC35 is another member

of the SR protein family, but SC35 has only one RRM and differs from SF2/ASF in RNA

binding specificity (Figure 4) (7,12,23,34,47,51,52). To rule out that the SR protein

inhibition of EIAV replication was due to an excess of SR proteins, we co-transfected the

EIAV molecular clone with either pSF2/ASF or pSC35. As before, co-transfection with

pSF2/ASF resulted in a significant reduction in virus titer (Figure 7A). SC35 did inhibit

EIAV replication, but the reduction was less than 10% of the inhibition observed with

SF2/ASF (6-fold verses 68-fold) (Figure 7A). Western blot analyses of cells transfected with

pSF2/ASF or pSC35 showed increased expression of both SR proteins above constitutive

levels (data not shown). Thus, while some inhibition of virus replication might be attributed

to excess SR protein, it appeared that the primary effect of SF2/ASF on EIAV replication

was specific to SF2/ASF and correlated with the RNA binding specificity of the SR protein.

To determine if the inhibition of EIAV replication was due to the inhibition of Rev

activity, we used an in vitro system that requires the presence of Rev in trans to produce

virus. Cf2Th/51 is a stably transfected cell line containing a Rev-defective pro virus, and RT

activity is produced only in the presence of Rev (3). When Cf2Th/51 cells were trans-

complemented with AERev and SF2/ASF, there was nearly a 3-fold decrease in supernatant

RT activity (Figure 7B). In contrast, when AERev was expressed in Cf2Th/51 cells with

SC35, there was only a modest 20% reduction in supernatant RT activity. Thus, the marked

inhibition of EIAV replication appeared to be due to a SF2/ASF-specific inhibition of Rev

activity. Together, the results further support a model wherein SF2/ASF inhibits EIAV

replication by competing with Rev for binding viral mRNAs and inhibiting Rev function.

Discussion

One function of SR proteins is to bind exon splicing enhancers (ESEs) and enhance

splice site recognition and exon inclusion in pre-mRNA splicing. The lenti viral protein Rev

binds the Rev responsive element (RRE) and mediates the nuclear export of unspliced and

incompletely spliced viral mRNAs, and is necessary for viral replication. In EIAV, the first

exon of rev contains a purine-rich sequence that functions as both a ESE and as a RRE

(ESE/RRE) (4,21). The ESE/RRE binds both SF2/ASF and ERev (4,13,21), and ERev

inhibits exon inclusion in in vitro splicing assays (4). In the present study, we examined

SF2/ASF inhibition of ERev-dependent nuclear export activity. SF2/ASF did not inhibit the

expression of Rev protein, but caused a dose-dependent decrease in Rev-dependent nuclear

export activity and EIAV replication. Rev activity could be restored by increasing

concentrations of pAERev, suggesting that SF2/ASF and Rev compete for some limiting

factor in the Rev-dependent nuclear export pathway. Deletion analyses indicated that the

RRM1/RRM2 of SF2/ASF was both necessary and sufficient to inhibit Rev activity and

EIAV replication. There was correlation between RNA binding specificity of SF2/ASF and

Rev inhibition, which suggested that SF2/ASF binding viral mRNAs inhibits both Rev

activity and virus replication. Together, these results support a model wherein SF2/ASF and

Rev compete for binding to viral mRNAs at the ESE/RRE. Therefore, factors that modulate

intracellular concentrations of SF2/ASF may be important in the regulation of EIAV Rev-

dependent nuclear export activity and virus replication.

The primary function of an SR protein RRM is to determine substrate specificity by

binding RNA in a sequence specific manner (12,34,49,51). Our data indicate that SF2/ASF

binding the ESE/RRE is required for the inhibition of Rev-dependent nuclear export activity

and EIAV replication, and suggest that SF2/ASF competes with Rev in Rev-dependent

nuclear export. Together, this supports a model wherein SF2/ASF competes with Rev for

binding the ESE/RRE. However, it remains unclear if the binding of SF2/ASF inhibits the

binding of Rev, or just Rev-dependent nuclear export activity. Rev binding to the ESE/RRE

inhibits ESE-dependent EIAV splicing (4,21). Therefore, it appears that binding the

ESE/RRE by either Rev or SF2/ASF inhibits the function of the other protein. Thus, it

remains to be determined if the SF2/ASF and Rev functional competition is the result of the

binding competition. Further studies must be done to elucidate the mechanism by which

SF2/ASF and Rev bind the ESE/RRE and to better understand the role of SF2/ASF in rev

exon 1 inclusion during splicing.

There are three, nearly consecutive AAAGAAGAA sequence motifs in the ESE/RRE

that mediate Rev binding, Rev-dependent nuclear export, SF2/ASF binding, and in vitro

splicing (Table 2) (4,13). SF2/ASF can bind RNA that contains either one, two, or three

motifs (13), whereas the two 3' motifs of the ESE/RRE appear to be necessary to promote

exon inclusion in an in vitro splicing assay (Table 2) (4). Interestingly, Rev binds RNA

containing either or both of the two 3' motifs, yet wild-type Rev-dependent nuclear export

activity depends on the presence of all three motifs (4). While it is not clear if both SF2/ASF

and Rev bind the exact same sequences, it is apparent that binding of one inhibits function of

the other. Chung and Derse have suggested that the ESE and RRE sequences are distinct, yet

overlap, and they have shown that Rev and SF2/ASF can bind the ESE/RRE simultaneously

(13). This suggests that the requirements for binding may be less stringent than the

requirements for functional activity, as data presented here suggested that Rev and SF2/ASF

are functionally competitive. If Rev nuclear export activity is dependent on presence of all

three motifs, it is possible that SF2/ASF binding at any one of the ESE/RRE motifs would

inhibit Rev nuclear export activity, but not binding. Further investigations are necessary to

characterize the specific interactions among SF2/ASF, Rev and the ESE/RRE that are

required for exon inclusion and nuclear export.

The SR proteins play a significant role in the regulation of gene expression in a

number of viruses. In adenovirus replication, the early and late phases of transcript

expression are regulated by the phosphorylation state of the SR proteins in infected cells

(24,26,36). Early transcripts express the viral protein E4-ORF4, which preferentially

interacts with hyperphosphorylated SR proteins as well as the cellular protein phosphatase

PP2A (24). The interactions among E4-ORF4, SR proteins, and PP2A induce the

dephosphorylation and inactivation of the SR proteins (6,36), which shifts the phase of

transcript expression from early to late. Thus, exogenous, hyperphosphorylated SR proteins

added during the early phase of mRNA expression inhibit the shift to the late phase and

subsequently, inhibit virus replication (35). In the lentivirus HIV, SF2/ASF has been

reported to bind the HIV RRE in a Rev-dependent manner and inhibit both Rev nuclear

export activity and replication (41). The HTV RRE is neither an ESE nor is it located with

the Rev gene, however, the RRM1/RRM2 of SF2/ASF was sufficient to inhibit HIV Rev

69

activity (40,41). Other HIV studies suggest that there may be a number of other proteins that

interact with SF2/ASF, HIV Rev, and the HIV RNA to regulate Rev activity and virus

replication (31,39,40,50). The finding that SF2/ASF can modulate EIAV Rev activity

provides an additional example of how viruses utilize complex cellular mechanisms of gene

regulation to regulate viral gene expression.

SR protein expression varies in different tissues and cellular activation states, which

can result in the expression of alternatively spliced mRNAs in a tissue or cell-specific

manner (22,43,53). For example, SF2/ASF expression increases in T lymphocytes in

response to activation, resulting in the alternative splicing of CD45 mRNAs (25). Our results

show that SF2/ASF inhibits EIAV replication in a dose-dependent manner. EIAV replicates

in monocytes/macrophages, and we have previously shown that activated and infected

macrophages impair viral replication through an, as yet, unidentified post-transcriptional

mechanism (44). It is possible that the activation of macrophages increases the cellular

expression of SF2/ASF, thereby decreasing viral replication by binding viral RNAs at the

ESE/RRE and inhibiting Rev activity. Indeed, SR protein expression varies during

development, differentiation, and proliferation (15). Further investigations to examine

intracellular concentrations of SF2/ASF during EIAV infection may identify a role for

SF2/ASF in regulating EIAV replication in vivo.

70

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26. Leppard, K.N. 1998. Regulated RNA processing and RNA transport during

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27. Liu, H.X., S.L. Chew, L. Cartegni, M.Q. Zhang, and A.R. Krainer. 1999. Exonic

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Cell. Biol. 20:1063-1071.

28. Liu, H.-X., M. Zhang, and A. Krainer. 1998. Identification of functional exonic

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29. Longman, D., I.L. Johnstone, and J.F. Carceres. 2000. Functional characterization

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30. Lopato, S., M. Kalyna, S. Dorner, R. Kobayashi, A.R. Krainer, and A. Barta.

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regulates splicing of specific plant genes. Genes Dev. 13:987-1001.

31. Luo, Y., H. Yu, and B.M. Peterlin. 1994. Cellular protein modulates effects of

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32. Mancuso, V.A., T.J. Hope, L. Zhu, D. Derse, T. Phillips, and T.G. Parslow. 1994.

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virus and equine infectious anemia virus. J. Virol. 68:1998-2001.

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10:1569-1579.

34. Mayeda, A., G.R. Screaton, S.D. Chandler, X.D. Fu, and A.R. Krainer. 1999.

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RNA recognition motifs and composite pre-mRNA exonic elements. Mol. Cell. Biol.

19:1853-1863.

35. Molin, M. and G. Akusjarvi. 2000. Overexpression of essential splicing factor

ASF/SF2 blocks the temporal shift in adenovirus pre-mRNA splicing and reduces

virus progeny formation. J. of Virology 74:9002-9009.

36. Nilsson, C.E., S. Perterson-Mahrt, A.R. Krainer, T. Kleinberger, and G.

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37. Olsen, H., A. Cochrane, P. Dillon, C. Nalin, and C. Rosen. 1990. Interaction of the

human immunodeficiency virus type 1 rev protein with a structured region in env

mRNA is dependent on multimer formation mediated through a basic stretch of

amino acids. Genes Dev. 4:1357-1364.

38. Payne, S.L., J. Rausch, K. Rushlow, R.C. Montelaro, C. Issel, M. Flaherty, S.

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1024.

40. Pongoski, J., K. Asai, and A. Cochrane. 2002. Positive and negitive modulation of

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978.

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42. Schaal, T.D. and T. Maniatis. 1999. Multiple distinct splicing enhancers in the

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The EMBO J. 20:1785-1796.

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48. Tacke, R. and J.L. Manley. 1995. The human splicing factors ASF/SF2 and SC35

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3551.

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79

55. Zapp, M L. and M R. Green. 1989. Sequence-specific RNA binding by the HIV-1

Rev protein. Nature 342:714-716.

Table 1. Summary of Rev. SF2/ASF. and ESE/RRE interactions.

RNA Motifs of the ESE/RRE RNA Functional Activities

1 2 3 Reva

Binding Nuclearb

Export SF2/ASF0

Binding In Vitrod

Splicing

-AAAGAAGAATCTAAAGAAGAAAAAAGAAGAA +++e +++ +++ +++

AAAGAAGAA +/- ++ ++ +/-

AAAGAAGAAAAAAGAAGAA +++ - +++ ++

AAAGAAGAA AAAGAAGAA ++ + ND +

a RNA binding studies were performed using Rev and the ESE/RRE as a substrate in

previous studies (4). The symbols indicate the binding activity of the motif(s).

b Transient Rev-dependent nuclear export assays were performed with Rev reporter plasmids

containing the ESE/RRE in previous studies (4). The symbols indicate the Rev-dependent

nuclear export activity of the motif(s).

c RNA binding studies were performed in previous studies using SF2/ASF and the RNA

sequences listed as substrates (13). The symbols indicate the ability of the motif(s) to bind

SF2/ASF. ND indicates not determined.

d In vitro splicing assays were performed in previous studies with RNA substrates containing

the motif listed (4). The symbols indicate the effect of the motif(s) on splicing of the

substrate.

e The activity of the RNA motifs on protein binding, nuclear export, or in vitro splicing

activity of the RNA motifs are indicated by symbols representing a gradation of effects from

no activity (-) to highest activity (+++).

80

Figure Legends

Figure 1. The EIAV genome, mRNAs, proteins, and rev exon 1 sequence. (A) The EIAV

genome, the mRNAs produced during replication, and the proteins they encode. The boxes

indicate the open reading frames. The horizontal, thick lines indicate the exon sequences of

the mRNA, and the diagonal thin lines indicate the spliced out RNA sequence. Messenger

RNA1 is a bicistronic, four exon mRNA that encodes both Tat (?) and Rev (r). Messenger

RNA2 is similar to mRNAl, but lacks rev exon 1 and encodes only Tat. The structural genes

of EIAV, including gag, pol, and env, are encoded by the singly spliced mRNA3 and the

unspliced mRNA4. (B) The sequence of rev exon 1. The Rev start codon is underlined, and

the light grey box indicates the identified EIAV RRE (light grey box)(4). The dark gray

boxes indicate the purine-rich sequences that function in both Rev binding, SF2/ASF

binding, Rev-mediated nuclear export, and ESE function. SA indicates the upstream splice

acceptor and SD indicates the downstream splice donor of the exon.

Figure 2. Characterization of AERev expression and activity. (A) Western blot analysis of

AERev expression in the presence of increasing concentrations of SF2/ASF. The bar graph

represents the counts of AERev obtained from phosphorimager analysis of the blot shown.

(B) Comparison of Rev nuclear export activity between wild-type Rev (white bars) and

AERev (gray bars). Results represent the mean of two independent transfections for wild-

type pERev and five independent transfections for pAERev. The error bars of the figure and

subsequent figures represent the standard error of the mean. Asterisks indicate the

concentration at which there is a significant difference in nuclear export activity between

wild-type Rev and AERev (PcO.Ol).

Figure 3. SF2/ASF inhibits Rev-dependent nuclear export activity. (A) Cf2Th cells were co-

transfected with 1 |lg pERRE-lA and increasing concentrations of pSF2/ASF in the presence

(—) or absence (—) of 50 ng pAERev. The results represent the mean value of at least six

independent transfections in the presence of pAERev and three independent transfections in

the absence of pAERev. The asterisk indicates the concentration at which there was no

significant reduction of Rev nuclear export activity as compared to cells transfected in the

absence of pSF2/ASF (P>0.05). All other concentrations of pSF2/ASF above 1 ng resulted

in a significant reduction of Rev nuclear export activity as compared to cells transfected in

the absence of pSF2/ASF (P<0.05). (B) Cf2Th cells were co-transfected with 1 |Lig pERRE-

1 A, 50 ng pSF2/ASF, and increasing concentrations of pAERev. The results represent the

mean of at least three independent transfections. All concentrations above 0 ng pAERev

resulted in a significant difference from cells transfected in the absence of pAERev (P<0.05).

Figure 4. Consensus RNA sequences bound by SR protein RRMs. SELEX analyses of the

individual RRMs of SF2/ASF and SC35 result in a number of RNA sequences that both SR

proteins will bind. The consensus sequence motifs of those RNA sequences are listed. The

symbols for alternative bases are: R=A/G, S=C/G, M=A/C, W=A/U, Y=C/U, and K=G/U.

The SF2/ASF consensus RNA binding motif RGAAGAAC nearly matches the three

AGAAGAA motifs in the ESE/RRE.

Figure 5. RRM1/RRM2 of SF2/ASF is necessary and sufficient to inhibit Rev-dependent

nuclear export activity. (A) SF2/ASF consists of two amino-terminal RRMs and a carboxy-

terminal RS domain. SF2/ASF deletion mutants were constructed and lack the RS domain or

one or both of the RRMs. (B) Cf2Th cells were co-transfected with 1 fig of pERRE-lA, 50

ng of p AERev (if indicated) and 50 ng of SF2/ASF construct. Results represent the mean

value of at least six independent transfections and are expressed as a percentage of the

AERev activity. The asterisks indicate significant differences from cells co-transfected with

only pERRE-lA and pAERev (f<0.001). There is no significant difference between values

from transfections with either pSF2/ASF or pSF2/ARS (P>0.05).

Figure 6. SF2/ASF and RRM1/RRM2 inhibit EIAV replication. (A) 2 jig of either

pSF2/ASF (—) or pcDNA3 (—) were co-transfected into cells with the EIAV infectious

molecular clone pSPEIAV-19R. (A) Supernatant RT activity over 48 hours. The results

represent the mean of six independent transfections as determined by phosphorimager

analyses of RT assays. (B) Infectious virus collected at 48 hours after co-transfection with 2

fig of pSPEIAV-19R and increasing concentrations of pSF2/ASF was titrated in equine

dermal (ED) cells. The results represent the mean of three independent transfections from

one of two experiments and are reported as focus forming units per mL of clarified

supernatant (FFU/mL). Values from all three concentrations of pSF2/ASF were significantly

different from control transfections with only pSPEIAV-19R (PcO.OOOl). (C) 1 fig of each

SF2/ASF expression construct was co-transfected into cells with 2 fig of pSPEIAV-19R, and

48 hours post-transfection clarified supematants were titrated for infectious virus on ED

cells. The results represent the mean of three independent transfections from one of two

experiments. The asterisks represent values significantly different from cells transfected with

only pSPEIAV-19R (P<108). There is no significant difference between values from

transfections with either pSF2/ASF or pSF2ARS (P>0.05).

83

Figure 7. SF2/ASF inhibition of EIAV replication is due to a specific inhibition of Rev

activity. (A) Cf2Th cells were co-transfected with 2 (ig of pSPEIAV-19R and 1 jxg of either

pcDNA3, pSF2/ASF, or pSC35. At 48 hours post-transfection, clarified supematants were

assayed for infectious virus. The results represent the mean of three independent

transfections from one of two experiments, and the error bars represent the standard error of

the mean. The asterisks represent values significantly different from cells transfected with

only pSPEIAV-19R (PcO.OOl) (B) Cf2Th/51 cells were co-transfected with 100 ng of

pAERev and 1 |ig of either pcDNA3, pSF2/ASF, or pSC35. 48 hours post-transfection,

clarified supematants were assayed for RT activity, and quantified by phosphorimager

analysis. The results represent the mean of at least six independent transfections. The

asterisks indicate a significant difference in values from cell transfected with pcDNA3

(f<0.01).

84

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87

SR Protein Domain(s) RNA Binding Motif

SF2/ASF RRM1/RRM2 RGAAGAAC AGGACRRAGC CRSMSGW

RRM1 ACGCGCA

RRM2 ND

SC35 RRM AGSAGAGUA GUUCGAGUA UGUUCSAGWU GWUWCCUGCUA GGGUAUGCUG GAGCAGUAGKS AGGAGAU GRYYMCYR UGCYGYY

Figure 4.

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91

CHAPTER 4. GENERAL CONCLUSIONS

Retroviruses utilize the post-transcriptional mechanism of splicing to produce the

necessary viral mRNAs for replication. Splicing during replication of simple retroviruses

produces only a singly spliced viral mRNA. Splicing during replication of complex

retroviruses, such as HTV, may produce more than 30 multiply spliced viral mRNAs (6).

Splicing in EIAV replication results in four major mRNA transcripts. As with other

lentiviral Rev proteins, EIAV Rev binds and exports the unspliced and singly spliced viral

mRNAs into the cytoplasm. Because Rev is absolutely required for virus replication, factors

that can inhibit Rev function would be expected to inhibit virus replication. EIAV produces

a persistent, life-long infection, and most infected animals show no sign of infection. Unlike

other lentiviral infections, which result in a chronic disease course, EIAV infection results in

a variable disease course, including periods of acute, chronic and/or inapparent disease. The

acute and chronic disease courses are characterized by periods of high virus replication

associated with cycles of fever, thrombocytopenia, and/or anemia (7,8). Therefore, virus and

host cell factors that affect virus replication, may also play a role in EIAV pathogenesis.

Alternatively Spliced Tat Transcripts

Splicing of the EIAV primary transcript produces, among other transcripts, a four-

exon, bicistronic mRNA that encodes Tat and Rev. The presence of Rev results in exon

exclusion and the subsequent expression of an alternatively spliced mRNA similar to four-

exon mRNA, but lacking exon 3. Exon 3 is also rev exon 1, and thus, Rev mediates

production of mRNAs that only encode Tat. Interestingly, the Tat start codon is a CUG, and

only by leaky scanning through the CUG is translation initiated at the Rev start codon, which

is the first AUG of the bicistronic message (2). Though the presence of a non-AUG start

codon upstream of an AUG start codon of a bicistronic message is not uncommon, the fact

that the start codon is a CUG and the fact that is the transcript is viral makes the bicistronic

EIAV mRNA uncommon.

We have found that the bicistronic and monocistronic Tat transcripts have the same

Tat activity, suggesting that the RNA sequences of exon 3 do not affect translation initiation

at the Tat CUG (Chapter 2). Our results also suggest that initiation at the Rev AUG and the

RRE of exon 3 do not have an effect on Tat translation. This is consistent with the

mechanism of leaky scanning, as Kozak states that translation initiation of the first start

codon is not influenced by a better initiation site downstream (5). Thus, the findings from

this work indicate that exclusion of exon 3 is not a mechanism to directly regulate Tat

expression, but may be a mechanism to produce Tat without producing more Rev.

SF2/ASF Inhibits EIAV Rev-mediated Nuclear Export and EIAV Replication

Interestingly, rev exon 1 contains a sequence that is both an ESE and an RRE

(ESE/RRE). This is unique among the lentiviruses as other lentiviral RREs are not ESEs and

are not located within RNA that encodes Rev. Though the ESE/RRE binds the SR protein

called SF2/ASF as well as Rev, the role of each in exon 3 inclusion/exclusion is not fully

described (1,3). Earlier work established that Rev inhibits ESE activity (1,4). We have

found that SF2/ASF inhibits Rev nuclear export activity in a dose-dependent manner, but not

by inhibiting Rev expression (Chapter 3) (1). Further, Rev and SF2/ASF appear to

functionally compete with one another.

As might be expected of a protein that inhibits Rev function, SF2/ASF also inhibited

EIAV replication. In fact, SF2/ASF potently inhibited EIAV replication. The inhibition of

both Rev nuclear export activity and EIAV replication mapped to the RNA binding domain,

made up of both RNA recognition motifs of SF2/ASF. These results suggest that SF2/ASF

and Rev may compete for binding the ESE/RRE. Because functional activity is dependent

on binding for Rev and typically so for SF2/ASF, a model of competitive binding would

support the in vitro functional competition observed.

Viruses do interact with SR proteins in many ways, and can exploit many of their

functions, which may influence both virus replication and host cells. Interestingly, the

impact of viruses on host cell gene expression through the exploitation of the SR proteins has

not been studied and may be an important area to investigate. Our results suggest that Rev

and SF2/ASF compete for binding the ESE/RRE, and it is possible that Rev and SF2/ASF

bind the same sequences. If so, Rev may bind cellular RNA sequences that are normally

bound by SF2/ASF, and could potentially affect cellular gene expression.

Future studies

The studies of this dissertation biologically characterize the effects of the interactions

of EIAV with Rev and SF2/ASF. The identification of SF2/ASF as a potent inhibitor of

virus replication in vitro suggests that the protein may be a cellular factor involved in EIAV

pathogenesis. There are a number of questions that need to be answered as to the role of

SF2/ASF in EIAV replication. Though we show that SF2/ASF inhibited both Rev nuclear

export activity and EIAV replication, we have not directly tested if the inhibition of EIAV

replication is due to an inhibition of Rev nuclear export activity. Therefore, future studies

should address this through Northern analysis of cytoplasmic and nuclear viral RNAs in the

presence of SF2/ASF.

From the research of this dissertation, the most logical, next step is to characterize the

specific interactions of Rev and SF2/ASF with the ESE/RRE. Only a few mutations have

been made in the ESE/RRE to investigate the RNA requirements for protein binding.

Therefore, purification of both Rev and SF2/ASF and their use in RNA gel shift assays will

determine if Rev and SF2/ASF compete for binding the ESE/RRE. A variety of RNA

substrates should also be used to determine if the ESE/RRE has important secondary

structure for protein binding. The results of these assays will assist in determining the

mechanism behind the functional competition apparent between Rev and SF2/ASF. I and

others have put a major effort into the purification of Rev protein for use in the assays, and

the protocol to purify Rev is nearly complete. Therefore, the purification of Rev should also

be pursued in the future, and the successful purification of functional Rev will be necessary

to directly test the hypothesis that Rev competes with SF2/ASF for binding the ESE/RRE.

In addition, the exact role of SF2/ASF in the activity of the ESE of exon 3 is

unknown, and future work should determine the role of SF2/ASF in exon 3 inclusion.

SF2/ASF binds the ESE in in vitro binding assays, but purified SF2/ASF has never been

used. The protein used in binding assays was from purified, total SR proteins (3). In

addition, in vitro splicing assays that have shown the Rev inhibition of ESE function have

always used purified, total SR proteins (1). Therefore, it will be important to use purified

SF2/ASF in in vitro splicing assays to determine if SF2/ASF is sufficient to promote exon 3

inclusion, and if purified Rev can inhibit exon inclusion.

95

It will also be important to determine if the SF2/ASF inhibition of EIAV replication is

biologically significant in in vivo virus replication. Though SF2/ASF expression has been

shown to vary among different tissues and cellular states, the levels of cellular expression

during replication and/or during different stages of disease are not known. Therefore, the

examination of SF2/ASF expression in cells before and after virus infection will assist in

determining if endogenous levels of SF2/ASF affect EIAV replication.

References

1. Belshan, M., G.S. Park, P. Bilodeau, C M. Stoltzfus, and S. Carpenter. 2000.

Binding of equine infectious anemia virus Rev to an exon splicing enhancer mediates

alternative splicing and nuclear export of viral mRNAs. Mol. Cell. Biol. 20:3550-

3557.

2. Carroll, R. and D. Derse. 1993. Translation of equine infectious anemia virus

bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation

codon. J. Virol. 67:1433-1440.

3. Chung, H.-K. and D. Derse. 2001. Binding sites for Rev and ASF/SF2 map to a 55-

nucleotide purine-rich exonic element in equine infectious anemia virus RNA. J.

BioLChem. 276:18960-18967.

4. Gontarek, R.R. and D. Derse. 1996. Interactions among SR proteins, an exonic

splicing enhancer, and a lentivirus rev protein regulate alternative splicing. Mol. Cell.

Biol. 16:2325-2331.

96

5. Kozak, M. 2002. Pushing the limits of the scanning mechanism for initiation of

translation. Gene 299:1-34.

6. Pollard, V.W. and M.H. Malim. 1998. The HtV-1 REV protein. Annu. Rev.

Microbiol. 52:491-532.

7. Sellon, D.C. 1993. Equine infectious anemia. Vet. Clin. North Am. Equine Pract.

9:321-336.

8. Sellon, D.C., F.J. Fuller, and T.C. McGuire. 1994. The immunopathogenesis of

equine infectious anemia virus. Virus Res. 32:111-138.

97

APPENDIX A. THE PURIFICATION OF EIAV REV

Introduction

Members of the lentivirus subfamily of retroviruses produce viral proteins during

replication that function in regulating the expression of viral mRNAs. The two most

common regulatory proteins are called Tat and Rev. Where Tat assists RNA polymerase II

in elongation to up-regulate viral transcription, Rev is an essential protein that functions by

localizing in the nucleus, binding unspliced or incompletely spliced viral mRNAs at a

nucleotide sequence called the Rev responsive element (RRE), and exporting the RNAs to

the cytoplasm through the exportin 1 pathway of nuclear export (reviewed in 4). The

structural proteins necessary for replication are encoded by the unspliced and incompletely

spliced viral mRNAs, and in the absence of Rev, these mRNAs are not expressed in the

cytoplasm. Thus, Rev is absolutely required for virus replication.

The lenti virus equine infectious anemia virus (EIAV) produces a Rev protein from

the third and fourth exons of a bicistronic, four-exon mRNA. Interestingly, rev exon 1

contains an EIAV RRE and binds Rev. The sequences of the RRE also function as an exon

splicing enhancer (ESE), which is necessary in the recognition and inclusion of rev exon 1

during splicing. In the presence of Rev, an alternatively spliced mRNA is produced identical

to the four-exon mRNA except lacking rev exon 1. The mechanism that results in Rev-

mediated alternative splicing has not been fully elucidated. Previous studies suggest that the

exclusion of rev exon 1 is due to competition between Rev and the cellular splicing protein

SF2/ASF for binding the sequences of the ESE and RRE. However, the exact sequences

bound by either protein are not known, and it appears they are not the same, but overlap

(ESE/RRE). Therefore, it was of interest to directly test the hypothesis that SF2/ASF and

Rev compete for binding the sequences of the ESE/RRE.

The 165 amino acid EIAV Rev protein (Figure 1) is functionally homologous to other

lenti viral Revs, but has little amino acid homology to them. Many of the functional domains

of EIAV Rev have yet to be fully defined, and those that have been mapped, are in different

locations of the protein than in other lenti viral Revs. For example, the amino acids

responsible for the nuclear export activity of the Rev (NES) are located toward the amino-

terminus of EIAV Rev (amino acids 32-55), whereas in other lenti viral Revs, the NES is

located near the carboxy-terminus. In addition, in vitro studies in our laboratory describe a

role for EIAV Rev amino acids 75-127 in alternative splicing (3), as scanning deletion

mutagenesis within amino acids 75-127 reduce Rev-mediated alternative splicing. Because

Rev and the SF2/ASF bind the ESE/RRE region, we hypothesize that the Rev alternative

splicing domain may be the RNA binding domain. To further define the functional domains

of EIAV Rev, it was of interest to map the RNA binding domain. By better understanding

how Rev and SF2/ASF bind the ESE/RRE, and by mapping the of the Rev RNA binding

domain, the role of Rev in EIAV alternative splicing will be better understood. Therefore, to

further characterize the functional role of Rev in EIAV alternative splicing, the first specific

aim was to purify functional EIAV Rev. Purification of Rev would result in a protocol for

use in the additional purification of a number of Rev deletion mutants, which could be used

in RNA binding assays to map the RNA binding domain.

99

Methods and Results

Glutathione S-transferase (GST) Gene Fusion System. Initial studies to purify

Rev utilized a prokaryotic GST-fusion protein expression system from Pharmacia Biotech.

cDNA fragments of an EIAV Rev clone (H21) (1) and a H21 Rev-derived mutant lacking

amino acids 32-55 (RevANES) were cloned into two vectors, pGEX-2T and pGEX-3X

(Amersham Biosciences), using the flanking 5' BamHl and 3' EcoRl sites. Each resulting

vector was transformed into both BL21 (Novagen, Madison, WI) and JM109 (New England

Biolabs, Beverly, MA) E. coli strains. The pGEX-2T vector encodes a GST protein upstream

of Rev with a six amino acid thrombin protease cleavage site immediately upstream of the

multiple cloning site of the vector. The pGEX~3X vector encodes a GST protein upstream of

Rev with four amino acids between the two proteins that are a Factor Xa protease cleavage

site. GST-Rev RNA transcription is induced from the tac promoter by addition of IPTG to a

bacterial culture. In protein purification, after inducing expression of GST-Rev, the bacteria

are harvested and lysed, and the lysate is added to a column containing a fixed glutathione

substrate, which binds to the GST portion of GST-Rev. Multiple washes remove the

unwanted proteins, and the purified fusion protein is eluted from the column with a reduced

glutathione buffer.

Initial studies optimized protein expression from the GST-H21 Rev plasmid;

examining a number of factors including the IPTG concentration, induction time, and the

method of lysis. Concentrations from 0.1 mM to 1 mM IPTG, induction times from one to

five hours, and lysis including sonication and the commercial buffer Bacterial Protein

Extraction Reagent (B-PER) (Pierce, Rockford, IL) were tested. Two different methods

using glutathione-fixed substrates were tested for binding, including a pre-packed column

100

(Pierce) and a batch purification (Amersham Biosciences). These experiments determined

that the optimal induction of GST-Rev was with 1 mM IPTG in log growth cells for 5 hours.

The best method for lysis was resuspension of the harvested pellet in B-PER. The best

purification was obtained by using the Glutathione Sepharose 4B bulk matrix. After

washing, GST-Rev protein was eluted with a 15 mM reduced glutathione buffer (50 mM

Tns-HCl, pH 8.0), dialyzed with TN buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0), and

quantified with a BCA assay (Pierce). Protein purification from bacteria consistently

resulted in low yields (-15 ng/ml bacterial culture) and degradation products. The GST-Rev

degradation products were used successfully in RNA binding assays and in in vitro splicing

assays to identify the EIAV RRE and ESE (Appendix B). Figure 2 shows a Western blot of

the protein products from the purification that were used in the experiments of the Appendix

B(2).

Because the goal of these studies was to purify Rev for use in RNA competitive

binding assays, a higher yield of pure protein was required. A variety of methods were

investigated to increase yield and decrease Rev degradation products. The first included the

use of the Rev ANES protein, which would eliminate a number of charged amino acids from

the protein that may have effect on induced GST-Rev expression (as Rev has always

empirically seemed poisonous to cells). A second variable tested was the bacterial cell type,

therefore, the protease deficient BL21 strain was used. The results indicated that the use of

Rev ANES protein did not improve yield, and that changing the host strain to BL21 did not

decrease the degradation.

In addition to other products, Rev appeared to cleave from GST-Rev in earlier studies

(Figure 2), and therefore, a number of studies were initiated to purify Rev from the degraded

101

sample. Initial studies with a size exclusion column did not result in purified Rev. However,

the results could have been due to the low yield, as no protein was detected by either protein

quantification assays or Western blot analysis. Therefore, to improve the yield of Rev from

the degraded preparation, protease cleavage of GST-Rev was investigated using the specific

protease sites between GST and Rev. Protease cleavage was performed on GST-Rev while

on the column, as well as on the eluted GST-Rev preparation. Results of protease cleavage

assays showed that Thrombin did not cleave GST-Rev, and Factor Xa cleaved GST-Rev at a

number of sites within Rev.

Rev purification using a baculovirus expression system. The goal of Rev protein

purification was to prepare purified Rev and Rev deletion constructs for use in RNA binding

assays. Studies of protein expression in a prokaryotic system resulted in low-yield and

degraded protein products. Protein purification was switched to a eukaryotic, baculovirus

expression system in hopes of increasing yield and purity, with an added benefit of expressed

protein that is post-translationally modified.

The MaxBac 2.0 Transfection and Expression kit was utilized (Invitrogen, Carlsbad,

CA). All kit protocols were used with the provided reagents, unless otherwise stated.

Briefly, a desired gene is cloned into the multiple cloning site of a baculovirus transfer vector

downstream of a sequence encoding six tandem histidine residues (6-His) and an

enterokinase cleavage (Xpress) tag. The transfer vector containing the desired gene is

transfected into insect cells with linearized Autographa californica multiple nuclear

polyhedrosis virus (AcMNPV) DNA. Through recombination, the transfer vector provides

the essential sequence needed for replication, including the desired gene. Recombinant virus

is selected, amplified, and when the virus infects cells, the desired protein is expressed.

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The H21 Rev cDNA was cloned into the baculovirus transfer vector pBlueBacHis2-C

(Invitrogen) using flanking 5' BamHl and 3' EcoRl restriction enzyme sites (pBBH-Rev21).

Standard kit protocols were followed to create a recombinant baculovirus that expresses Rev.

Linearized viral DNA (Bac-N-Blue DNA) was transfected with pBBH-Rev21 into the

Spodoptera frugiperda derived insect cell line Sf9. At 72 hours post-transfection,

supernatant was harvested and assayed for budded, recombinant virus by a viral plaque

assay. During the assay, 5-bromo-4-chloro-3-indolyl-f3-D-gaIactoside was added to the

growth media, and recombinant viral plaques appeared blue in color. Individual plaques

from the plaque assay were removed and placed in separate wells of a 12 well microliter

plate with 5 X 105 Sf9 cells/well resulting in a P-l stock. After 72 hours, viral DNA was

isolated, and PGR was performed with primers flanking the Rev gene to confirm recombinant

virus contained Rev. From individual PGR positive wells, 20 (0,1 of the P-l viral stock was

used to infect a 25 cm2 flask with 2X106 Sf9 cells to generate a high-titer P-2 viral stock.

From the P-2 stock a high-titer, large-volume P-3 stock was generated and titered for use in

Rev expression and purification studies.

Initial studies determined the optimal multiplicity of infection (MOI), expression time

post-inoculation, and preparation of cell lysate. In addition, Rev expression was confirmed

by Western blot analysis of infected cell lysates using polyclonal rabbit anti-Rev sera and a

mouse monoclonal antibody to the 6-His tag portion of the protein. The optimal MOI was 1

and the optimal expression time post-infection was 4 days. The 6-His Tag allowed protein

purification to be done in either native or denaturing conditions, and the Xpress System

Protein Purification kit (Invitrogen) was used. In both native and denaturing conditions, the

lysate was added to a column containing the nickel affinity resin ProBond (Invitrogen).

After multiple washes, the protein is eluted using either an imidazole buffer (3M imidazole,

500 mM NaCl, 20 mM NaP04, pH 6.0) for native elution, or a low pH buffer (8M Urea, 20

mM Na2HP04, 500 mM NaCl, pH 4.0) for denaturing elution.

Initial analyses of Rev purification using freeze/thaw for lysis in native conditions

showed that freeze/thaw did not release Rev into the lysate, and the majority of Rev was

located in an insoluble fraction. Therefore, we investigated other lysis methods to improve

the soluble fraction of Rev. Results of the studies showed that the addition of mild-

detergents could increase the amount of soluble Rev in the lysate, but the 6-His Rev did not

bind the column. Therefore, we next investigated purification of Rev in denaturing

conditions.

As per the denaturing conditions purification protocol, the cell pellets were

resuspended in 10 mis of Guanidinium Lysis Buffer. Cell lysates were passed through an 18

gauge needle four times and then centrifuged at 20,000 X g for 10 minutes. Supematants

were added to a pre-equilibrated ProBond resin column in two, 5 mis aliquots. After addition

of each aliquot, the resin was resuspended and the column was rocked for 10 minutes to

allow the protein to bind. The resin was then settled by centrifugation at 800 X g for 2

minutes, and the supernatant was removed. The column was washed twice with 4 mis of

Denaturing Binding Buffer, five times with 4 mis Denaturing Wash Buffer 6, and four times

with 4 mis of Denaturing Wash Buffer 5.3. Each wash cycle consists of resuspending the

resin in the wash solution, rocking the column for 2 minutes and settling the resin by

centrifugation at 800 Xg for 2 minutes. To confirm the efficacy of each wash, absorbance

values at 260 nm were monitored by a spectrophotometer. After washing, the protein was

eluted from the column, by adding 7 mis of Denaturing Elution Buffer and collecting 1 ml

104

aliquots. Protein aliquots were snap frozen in N2 (1) and stored at -80°C. Protein elution was

again monitored by spectrophotometer with absorbance values at 260 nm. Fractions that

contained protein were analyzed by Western blot analysis, and results showed that the

fractions contained pure Rev.

Large amounts (1 mg/ 1.0X106 cells) of pure Rev were successfully purified under

denaturing conditions (Figure 3). Because the protein was denatured, it is expected to have

no functional activity. Denatured Rev functional activity could not be tested, because the

denaturing buffer inhibited in vitro functional assays. Therefore, a number of studies were

performed to renature the protein, using both gradient and step-dialysis. Briefly, 8 M Urea

was added to a base buffer containing 20 mM Hepes, pH 7.9, 2.5 mM MgCl2, 1 M KC1, 1

mM EDTA. During the renaturation, protein dialysis occurred in either buffers with half

molar decreasing steps in Urea or in the base buffer containing 8M urea, and increasing

amounts of base buffer over time. In either gradient or step dialysis, the results showed that

Rev precipitated when the Urea concentration decreased below 3 M. To improve the

solubilization of Rev, further studies used buffer containing the base reagents and varying

concentrations of other reagents to reduce protein-protein interactions. These included: NaCl

(< 1 M), MgCl2 (< 10 mM), P-mercaptoethanol (BME)(<6 mM), DTT (0.5 mM), and tRNA

(-100 |ig/|il). The results showed some success, and the best result in which Rev stayed

soluble to 1 M Urea resulted from renaturation by step dialysis in a buffer containing a

variable concentration of Urea (6M to 1M) in 20 mM Hepes, pH 7.9, 1 M KC1, 10 mM

MgC12,1 mM EDTA, and 6 mM BME. The same result was also observed in other studies

using a low pH buffer (50 mM NaOAc, 1 M KC1, pH 5.0).

Recent studies have investigated the possibility of renaturation while on the nickel

column, and those studies showed that the protein was released from the column as the Urea

concentration decreased. Finally, studies have shown that renaturation of a Rev mutant

expressed by the baculovirus system in which a large number of charged amino acids were

deleted (A144-165), also precipitates at 1 M Urea. Future studies will be in purification of

Rev amino acids 75-127, as it our hypothesis that these amino acids contain the RNA binding

domain.

Acknowledgements

I would like to thank Michael Belshan and Sean Murphy, who initiated these studies

and cloned the Rev cDNA into pGEX-2T. In addition, I thank Yuanbin Ru for the most

recent studies of protein renaturation on the column and the renaturation attempt of the

RevA144-165.

References

1. Belshan, M., M.E. Harris, A.E. Shoemaker, T.J. Hope, and S. Carpenter. 1998.

Biological characterization of Rev variation in equine infectious anemia virus. J. Virol.

72:4421-4426.

2. Belshan, M., G.S. Park, P. Bilodeau, C M. Stoltzfus, and S. Carpenter. 2000.

Binding of equine infectious anemia virus Rev to an exon splicing enhancer mediates

alternative splicing and nuclear export of viral mRNAs. Mol. Cell. Biol. 20:3550-3557.

106

3. Murphy, S.M., M. Belshan, P. Bruellman, Y. Li, T.J. Hope, and S. Carpenter. In

preparation. Functional domains of equine infectious anemia virus Rev.

4. Pollard, V.W. and M.H. Malim. 1998. The HTV-1 REV protein. Annu. Rev.

Microbiol. 52:491-532.

Figure Legends

Figure 1. EIAV Rev amino acid sequence. The amino acid sequences of a cDNA isolated

from Th-1 infected horse macrophage cultures (1). NES indicates the identified nuclear

export signal (amino acids 32-55). RBD/ASD indicates the putative RNA binding

domain/alternative splicing domain (amino acids 75-125). NLS indicates the nuclear

localization signal (amino acids 160-165).

Figure 2. Bacterial expressed GST-Rev. GST-Rev has an estimated molecular weight of -46

kDa. GST has a estimated molecular weight of -29 kDa, and Rev has an estimated

molecular weight of -18 kDa. Convalescent horse serum was used for the detection of Rev,

and a monoclonal antibody against GST was used for the detection of GST. Uninduced cell

lysate and GST-induced cell lysate were run a separate blot and are not shown.

Figure 3. Baculovirus expressed 6-His Rev. Rev was nickel affinity purified. Anti-Xpress is

a mouse monoclonal antibody that recognizes an epitope within the enterokinase cleavage

site. The tagged Rev protein has an estimated molecular weight of 24 kDa. The primary Rev

detection reagent is an anti-Rev polyclonal sera made from Rev-peptide injected rabbits.

107

1 40 MAESKEARDQEMNLKEESKEEKRRNDWWKIDPQGPLESDQ

NES

41 80 WCRVLRQSLPEEKIPSQTCIARRHLGPGPTQHTPSRRDRW

NES RBD/ASD

81 120 IRGQILQAEVLQERLEWRIRGVQQVAKELGEVNRGIWREL

RBD/ÀSD 121 160

YFREDQRGDFSAWGGYQRAQERLWGEQSSPRVLRPGDSKR RBD ZASD NLS

161 165 RRKHL

NLS

Figure 1

108

KDa

" - 43.9- GST-Rev

- -32.1- *

Rev—• • -18.1-

anti-EIAV anti-GST

Figure 2

GST-Rev

Degraded / GST-Rev \

109

AcMNPV infected Sf9 lysate

wt Rev Rev wt kDa

- 32.5

6-His Rev —• on»

• 18.4

anti-Xpress anti-Rev

Figure 3

110

APPENDIX B. BINDING OF EQUINE INFECTIOUS ANEMIA VIRUS REV TO AN

EXON SPLICING ENHANCER MEDIATES ALTERNATIVE SPLICING AND

NUCLEAR EXPORT OF VIRAL MRNAS

A paper published in the journal Molecular and Cellular Biology. 2000. 20(10): 3550-3557.

Michael Belshan1, Gregory S. Park1, Patricia Bilodeau2, C. Martin Stoltzfus2,

and Susan Carpenter1*

Abstract

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs,

equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of

the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a

fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage

were correlated with sequences necessary for Rev binding and transport of incompletely

spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for

exon 3 recognition and the addition of Rev inhibited exon 3 splicing. GST-Rev bound to

probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited

both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These

results suggest that Rev regulates alternative splicing by binding at or near the ESE to block

'Department of Veterinary Microbiology and Preventive Medicine, Iowa State University,

Ames, IA, 50011, and ^Department of Microbiology, University of Iowa, Iowa City, IA,

52242

I l l

SR protein-ESE interactions. A 57 nt sequence containing the ESE was sufficient to mediate

Rev dependent nuclear export of incompletely spliced RNAs. Rev export activity was

significantly inhibited by mutation of the ESE, or by ^^-complementation with SF2/ASF.

These results indicate that the ESE functions as a Rev responsive element (RRE), and

demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions

required for efficient export of incompletely spliced viral RNAs.

Introduction

Retroviruses utilize a variety of mechanisms to differentially express numerous

proteins from relatively small genomes which possess a single transcriptional start site. These

mechanisms include, but are not limited to, the use of polyprotein precursors, ribosomal

frameshifting (19), alternate start codons (6), bicistronic mRNAs (6), and alternative splicing

(29). Alternative splicing allows for the production of multiple viral mRNAs from a single

RNA precursor. The simplest retroviruses, such as murine leukemia virus, express only two

mRNAs, an unspliced RNA which serves as both mRNA for the gag and pol proteins and as

progeny viral RNA, and a singly spliced mRNA which encodes the env gene. In contrast,

complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1) produce at

least 20 different mRNAs, including several multiply spliced RNAs that encode small

regulatory proteins (29).

In all retroviruses, alternative splicing requires the presence of suboptimal splice

sites, allowing for expression of several mRNAs from a single pre-RNA. Regulation of

suboptimal splice sites is a complex process mediated in part by cis-acting RNA sequences

that can either enhance or repress recognition of a splice site by the splicing machinery. Exon

splicing enhancers (ESEs) and silencers (ESSs) have been described for many virus and

cellular RNAs (2, 23). ESEs typically are purine rich sequences embedded within

alternatively spliced exons. The purine rich sequences mediate exon recognition through

interactions with members of the SR protein family of splicing factors. SR proteins are both

essential splicing factors and regulators of alternative splicing (reviewed in (16)). Binding of

SR proteins to an ESE recruits essential splicing factors to suboptimal splice sites near ESE

sequences, resulting in exon inclusion of alternatively spliced exons.

In addition to the above mechanisms, complex retroviruses utilize Rev-like pathways

to differentially regulate expression of incompletely spliced RNAs encoding for virion

structural and enzymatic proteins and progeny RNA molecules (reviewed in (9)). The

prototype member of this family, HTV-1 Rev, binds to the viral pre-mRNA at a specific

sequence called the Rev-responsive element (RRE) (8, 35), multimerizes (24, 34), and

facilitates export of incompletely spliced RNAs from the nucleus via a Crml-mediated

pathway (13, 14). Equine infectious anemia virus (EIAV) Rev is functionally homologous to

HIV-1 Rev (15), but is less well characterized. EIAV Rev is a 165 amino acid protein

translated from exons 3 and 4 of a multiply spliced, 4-exon, bicistronic mRNA which also

encodes the tams-activating protein, Tat (Fig. 1) (6). In addition to promoting nuclear export

of incompletely spliced RNA, EIAV Rev also regulates inclusion of exon 3 of the multiply

spliced RNA. In the presence of Rev, a multiply spliced mRNA lacking exon 3 is produced

(22). Rev variants which are NES-defective have been shown to mediate alternative splicing

(4, 18); however, it is not known if the alternative splicing function is required for nuclear

export activity. Exon 3 is flanked by a suboptimal splice acceptor and contains a purine rich,

exon splicing enhancer-like motif which has been shown to interact with the SR protein

113

SF2/ASF (17). Derse and co-workers have proposed that EIAV Rev-mediated skipping of

exon 3 is a consequence of Rev-RNA interactions which directly or indirectly inhibit

SF2/ASF (17). We previously mapped a RRE of Rev to a 534 nt region containing exon 3

(4), suggesting the possibility that Rev mediates exon 3 skipping by binding at or near the

purine rich sequence to disrupt SR protein interactions necessary for exon 3 recognition.

Here, we further delineate the role of Rev in exon 3 alternative splicing. Our results

indicate that the purine rich sequence in exon 3 is required for the utilization of the exon 3

splice acceptor, confirming the presence of an ESE within exon 3. RNA gel mobility shift

assays and nuclear export assays demonstrate that Rev binds to the ESE, and that this binding

facilitates RNA export. Together, these results indicate the exon 3 ESE is a RRE of EIAV.

Therefore, Rev mediates exon 3 alternative splicing by binding the viral pre-mRNA at the

ESE/RRE and interfering with SR protein - ESE interactions.

Materials and Methods

PCR and Plasmid construction. All plasmid constructs were confirmed by sequence

analysis (Iowa State University DNA Synthesis and Sequencing Facility). DNA templates for

splicing substrates were amplified from p33k, a subclone of the p26 EIAV proviral clone

previously described (5). Unless otherwise indicated, PCR reactions were performed as

directed by the manufacturer (Perkin Elmer, Foster City, CA) using 1 jU,M primers. Standard

PCR reactions consisted of 25 cycles of one minute denaturation at 94° C, one minute

annealing at 50°C, and one minute extension at 72° C, followed by an addition cycle with a

prolonged, five minute extension. All DNA templates for splicing substrates used a common

5' primer, CTGAAGGCAATCCAACAAGG; and individual 3' primers to generate the

114

substrates shown in figure 2A. The 3' primers used and the region of EIAV amplified were:

CTCTCTATGATAAGCTTC, EIAV nt 5233-5793; CCAGTAGTTCCTGCTAAGCA, nt

5233-5573; TTTCCACCAGTCATTTCTTC, nt 5233-5535; CAGGTTCATTTCTTGGTCT,

nt 5233-5490. All nucleotide numbering is based on that of Kawakami et al. (20). After PCR,

fragments were TA cloned into the pGEM-T easy vector as directed by the manufacturer

(Promega, Madison, WI).

The expression plasmid pRevWT was described previously as pcH21 (4). pDM138,

pERRE-All (EIAV nt 5280-7534), and pERRE-1 (nt 5280-5834) have also been previously

described (4). To construct pERRE-lA, primers containing a Clal restriction site were used

to amplify EIAV nt 5281-5795. ERRE-1A 5' primer:

GG ATCG ATTTGAT AT ATGGG ATT ATT: 3' primer:

GGATCGATCTCTCT ATG AT A AGCTTC (Clal sites are underlined). The minRRE

sequence (EIAV nt 5485-5540) was synthesized as complementary oligonucleotides with

Clal extensions on the 5' and 3' ends. The oligonucleotides were heated at 95° C for 5

minutes and annealed by slow cooling. The fragment was phosphorylated, then ligated with

pDM138. The pGST-Rev expression vector contains a cDNA cloned in frame into the

BamHl site of the GST fusion vector pGEX-3X (Amersham Pharmacia Biotech, Piscataway,

NJ).

ESE mutants were constructed by PCR-Ligation-PCR mutagenesis according to the

methods described by Ali et al. (1) using internal primers designed with the specified

mutations shown in figure 4A. The two regions were amplified with Vent DNA polymerase

(New England Biolabs, Beverly, MA). The 3' fragment was phosphorylated, ligated with the

5' fragment, and 2 fil of the ligation reaction was PCR amplified with outer primers described

115

above to amplify EIAV nt 5233-5793. Amplicons were cloned into pGEM-T. To construct

the mutant pDM138 constructs, the pERRE-lA primers described above were used to PCR

ampify the respective mutants from the pGEM-T background and clone into the Clal

restriction site of pDM138.

pSF2/ASF was generated from the PET9c-SF2 plasmid obtained from Dr. Adrian

Krainer, Cold Spring Harbor Laboratory (21). The cDNA region corresponding to SF2/ASF

was cloned as two fragments: a Bglll to Kpnl (-100 to +242; numbering based on +1 at

initiation AUG), and Kpnl to BamHl (+242 to a Bam HI site downstream of the UAA

terminator). These were inserted into the eukaryotic expression vector

pCMV5 (provided by Mark Stinski, University of Iowa) which was cleaved by

Bglll and BamHl.

Synthesis of RNA substrates. The plasmids containing the splicing substrates were

digested with Spe I (GIBCO BRL, Rockville, MD) to create linearized templates for

transcription of RNA splicing substrates. In vitro run-off RNA transcripts labeled with

[32p]urp (Amersham Pharmacia Biotech) were generated as previously described (2). DNA

templates for RNA binding analysis were amplified by PCR from p33k using 5' primers

containing a T7 promoter site (a diagram of the substrates is in Fig. 3A). The primers used

for the substrates were: 5' primer:

TAATACGACTCACTATAGGGAGGAACAGCATGGCAGAATCG. 3' primer:

TTTCCACCAGTCATTTCTTC (RREpl, nt 5443-5535); 5' primer:

T A AT ACG ACTC ACT AT AGGG AGGTG A A AGA AG A ATCT A A AG. 3' primer:

CCACCAAAGTATTCCTCC (RREp2, nt 5489-5589); 5' primer:

T AAT ACG ACTC ACT AT AGGGAGGTGACTGGTGGAAAATAGG.3 ' primer:

116

CCCT AT ATA ATGTTGCTG (RREp3, nt 5523-5622); 5' primer:

TA AT ACG ACTC ACT AT AGGG AGGCGG AGG A AGC AAGAG ACC, 3' primer:

CCTGCT A AGC AT A AC AG A (RREp4, nt 5458-5565). The T7 promoter is underlined in the

5' primers. Amplified DNA was phenol.chloroform extracted, ethanol precipitated, and

resuspended in RNase-free dH2Q. The RREp5 DNA fragment was synthesized as two

complementary oligonucleotides containing the T7 promoter attached to EIAV nt

5485-5540. Complementary DNA fragments were combined at equal molar amounts, heated

at 95° C for 5 minutes, then slow cooled to anneal.

Expression/Purification of GST-Rev. BL21 Escherichia coli transformed with the

pGST-Rev expression vector were grown overnight at 1/10 final culture volume in NZY

broth containing 0.1 mg/ml ampicillin. The next day, cells were brought up to final volume,

grown an additional 3 hours, then induced with 1 mM IPTG for 5 hours. After induction,

cells were washed 3 times and resuspended in 50 mM Tris (pH 8.0), 50 mM NaCl (TN

buffer). Cells were lysed by sonication and the supernatant clarified by centrifugation at

10,000 xg. GST-Rev was purified by binding to glutathione sepharose 4B beads (Amersham

Pharmacia Biotech) overnight and washed 3X with TN buffer. The fusion protein was eluted

with 15 mM reduced glutathione in 50 mM Tris, pH 8.0, concentrated with a 30 kDa MWCO

filter concentrator (Millipore, Bedford, MA), and dialyzed against TN buffer. Protein

expression was confirmed by SDS-PAGE and immunoblotting with convalescent anti-EIAV

antisera or anti-GST antisera (Amersham Pharmacia Biotech) which detected expression of

the GST-Rev fusion protein and several minor bands, including GST alone (data not shown).

In some cases, the fusion protein was digested with 4 units/bead bed volume of Factor Xa

protease (Amersham Pharmacia Biotech) while bound to glutathione sepharose beads. Excess

117

GST and GST-Rev fusion protein were removed with the glutathione sepharose beads, and

the supernatant, containing cut Rev protein, concentrated and dialyzed as described above.

In vitro splicing, and gel electrophoresis. Splicing reactions were carried out as

previously described (2). In brief, approximately 8 fmol of EIAV RNA substrates were

incubated for 2 hours at 30°C with 60% (v/v) nuclear extract in Dignam's buffer D (12), 20

mM creatine phosphate, 3mM MgCl%, 0.8 mM ATP and 2.6% (w/v) polyvinyl alcohol. In

some experiments, EIAV Rev protein was diluted in buffer D (12) and added to the splicing

reactions at the indicated concentrations. RNAs were analyzed on 4% polyacrylamide gels

containing 7 M urea.

RNA binding Assays and gel electrophoresis. RNA - protein interactions were

performed in IX RNA binding buffer containing 10 mM HEPES-KOH (pH 7.5), 100 mM

KC1, 1 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 50 ng/gl E. coli tRNA, and 10% glycerol.

RNA was in vitro transcribed in the presence of [32P]UTP as described above. 100 ng to 2 mg

of GST or GST-Rev fusion protein was incubated with approximately lxlO6 cpm RNA

probe on ice for 15 minutes. The reactions were directly loaded onto a 8% native 100 mM

tris-glycine polyacrylamide gel (37.5:1 acrylamiderbis cross-linking ratio) which had been

pre-run 1 hour. The samples were electrophoresed an additional 3 hours. The gel was fixed in

20% ethanol, 10% acetic acid for 15 minutes, dried, and exposed to X-ray film with an

intensifying screen.

CAT Assays. Transient transfections and CAT assays were performed using human

embryonic kidney (293) cells and canine fetal thymus (Cf2th) cells. Cells were maintained in

Dulbecco's modified eagle medium supplemented with 10% fetal calf serum and

penicillin/streptomycin. CAT assays using 293 cells were performed as previously described

118

(4). Briefly, 1 jug of either pcDNA3 (Invitrogen, Carlsbad, CA) or pRevWT was transfected

by calcium phosphate co-precipitation with 0.2 jig of pDM138 reporter plasmid, 0.2 jig of

pCHllO, and 0.6 gg of pUC19. Two days post transfection, cells were harvested,

resuspended in 0.3 ml 0.25 M Tris (pH 7.5), lysed by freeze/thawing, and assayed for (3-

galactosidase activity to normalize CAT assays for transfection efficiency. Normalized

lysates were assayed for CAT activity using 3 fû ["^chloramphenicol and 1 mM acetyl

CoA. Acetylated products were separated by thin layer chromatography and the percentage

acetylation was quantified by phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Experiments were performed in triplicate and results summarize a minimum of six

independent transfections.

Cf2th cells were used for in vivo competition assays. Cells were transfected with 0.5

ng pRevWT, 0.3 jig of pERRE-1 reporter plasmid, and ten-fold increasing concentrations (0-

100 ng) of pSF2/ASF using 4 jxl TransIT-LT reagent (Mirus Corporation, Madison, WI).

Each reaction also included 0.2 gg of pCHl 10 and pUC19 to equalize the total DNA per

reaction. Two days post-transfections, cells were harvested and lysates were assayed for (3-

galactosidase activity as above. Normalized lysates were assayed for CAT using a

commercially available CAT-ELISA kit (Roche Molecular Biochemicals).

Results

The purine rich sequence is required for exon 3 recognition. Exon 3 of the

bicistronic, four exon equine infectious anemia virus (EIAV) mRNA contains a purine rich

sequence which resembles an exon splicing enhancer (ESE). Previous reports showed that

the SR protein SF2/ASF cross-links to the ESE-like sequence in vitro and suggested the ESE-

119

like sequence may enhance exon 3 inclusion during pre-mKNA splicing (17). To further

investigate the ds-acting requirements for exon 3 inclusion, we constructed a series of DNA

templates to generate radiolabeled RNA substrates for in vitro splicing. All substrates

contained the exon 2 splice donor, the intervening intron, exon 3, and downstream sequences.

Nested 3' deletions were made to identify splicing enhancer sequences present within or

downstream of exon 3 (Fig. 2A). Splicing of radiolabeled substrates was assayed in vitro

using HeLa cell nuclear extracts, which include all SR proteins. All constructs containing the

purine rich sequence were spliced (Fig. 2B, lanes 3-5), whereas no splicing was observed

using the substrate lacking the purine rich sequence (Fig. 2B, lane 2). This is consistent with

the hypothesis that the purine sequence functions as an ESE and is required for exon 3

inclusion in the multiply spliced four exon transcript. Taken together with previous work

(17), this suggests exon 3 recognition requires SF2/ASF interactions at the ESE. The

addition of as little as 100 ng of Rev to a splicing reaction containing the largest splicing

substrate inhibited exon 3 recognition (Fig. 3). This confirms earlier, in vivo observations of

Rev-mediated changes of alternative splicing (17), and indicates that Rev is the only viral

protein necessary for exon 3 skipping.

Rev binds the ESE. Derse has suggested that binding of EIAV Rev to a region of

the viral pre-mRNA near the ESE results in either a direct or indirect inhibition of SR protein

function (17). In previous work, we identified an RRE region spanning exon 3 (nt 5280 to

5834) (4). This favors a mechanism wherein Rev-RRE interactions disrupt SF2/ASF binding

at the ESE. To examine whether Rev binds at or near the ESE, we generated a series of RNA

probes and tested for Rev-RNA interactions by RNA gel mobility shift assays using a

bacterially expressed and purified GST-Rev fusion protein. The location of the RNA probes

120

relative to exon 3 and the ESE is shown in Fig. 4A. GST-Rev bound to exon 3 probes RREpl

and RREp2, which both contain the ESE; however, no binding was observed with the GST

negative control (Fig. 4B, lanes 1 to 4). Minor binding was observed with RREp3 (Fig. 4B,

lane 6), which has sequences immediately downstream of the ESE (nt 5523 to 5622), but

lacks the purine rich region. The binding site was further delineated to a 57 nt region of viral

RNA using two smaller ESE-containing probes, RREp4 and RREp5. GST-Rev interacted

with both probes (Fig. 4B, lanes 8 and 11), further suggesting that Rev binds at or near the

ESE. To confirm the specificity of binding, gel shift assays were performed with RREp2 in

the presence of excess cold competitor RREp2 or RREp3 (Fig. 4C). Excess cold RREp2

inhibited GST-Rev binding (Fig. 4C, lanes 2 and 3), whereas no inhibition was observed with

RREp3 (Fig. 4C, lanes 4 and 5), demonstrating that the binding of GST-Rev to the ESE-

containing RREp2 is specific. No slower migrating bands, indicative of Rev multimerization,

were observed in any of the RNA binding analysis, although multimerization was readily

observed with HIV-1 Rev when used as a positive control (data not shown). The minor bands

in Fig. 4C represent GST-Rev and degraded by-products. Overall, these results demonstrate

that Rev specifically interacts with the viral RNA at or near the ESE.

Mutagenesis of the ESE reduces exon 3 splicing and Rev binding. The finding that

GST-Rev bound to a 57 nt region containing the ESE suggested that a Rev - RNA interaction

was directly competing with SF2/ASF for binding at the ESE. If so, there should be similar

sequence requirements for exon 3 recognition and Rev binding. The ESE contains two purine

rich sequences (designated A and B), which include seven GAA repeats (Fig. 5A). GAA

repeats have been shown to be important for SF2/ASF recognition of ESE sequences in other

systems (reviewed in 16). Therefore, we constructed five ESE mutants in the largest splicing

construct and the RREp4 RNA probe fragment which contained various GAA to GÇA

mutations (Fig. 5A). The mutant templates were tested for in vitro splicing and GST-Rev

binding. Mutation of all GAA motifs (mutAll) or the B purine stretch (mutB) resulted in a

decrease in both exon 3 in vitro splicing (figure 5B, lanes 3 and 5) and in GST-Rev binding

(figure 5C, lanes 2 and 4). Mutation of the GAA repeats in only the B purine stretch resulted

in a more modest reduction in both in vitro splicing and GST-Rev binding (Fig. 5B, lanes 6

and 7 and 5C, lanes 5 and 6). Mutation of GAA repeats in the A region (mutA) did not

appear to significantly affect either exon 3 splicing or GST-Rev binding in vitro (Fig. 5B,

lane 4 and 4C, lane 3), suggesting the B purine stretch alone contains cis-acting sequences

necessary for exon 3 recognition and GST-Rev binding. The finding that each mutant had

comparable effects in both assays suggests similar requirements in the ESE for both exon 3

recognition and Rev binding, further supporting a model of Rev inhibition of splicing

through direct competition with SR proteins for binding at the ESE.

The ESE can function as a RRE to mediate RNA nuclear export. In other

complex retroviruses, Rev functions to regulate the export of incompletely spliced RNAs via

interaction with the viral pre-mRNA at a specific sequence called the Rev responsive element

(RRE). We had previously used the HIV-1 derived pDM138 reporter system to preliminarily

map the RRE of EIAV to a region which overlapped exon 3 (ERRE-1) (4). However, this

fragment possessed only 52% of the activity of a reporter containing a much larger fragment

of EIAV (ERRE-All), suggesting further downstream sequences enhanced Rev-mediated

export. The RNA binding data given above suggested that the functional sequence within

ERRE-1 was the ESÉ sequence. To test this, we constructed a pDM138 reporter plasmid,

minRRE, which contains only 57 nt of EIAV (nt 5485-5540, RREp5), spanning the ESE and

122

the remainder of exon 3. Transient transfection assays in 293 cells demonstrated the minRRE

reporter produced levels of CAT activity comparable to ERRE-1 (Fig. 6A), but only 35% of

the activity ERRE-All. This indicates minRRE contains the functional RRE in ERRE-1,

however additional elements outside ERRE-1 may be required for full export activity.

To confirm the ESE is the RRE within minRRE, we introduced the GAA to GCA

mutations used for in vitro splicing and RNA binding assays (Fig. 5A) into a reporter vector

containing the same sequences present in the largest splicing substrate (Fig. 2A). This vector,

ERRE-1 A, is 41 nt shorter than ERRE-1, but exhibited similar levels of activity as ERRE-1

(Fig. 6A). In all cases, mutation of the GAA repeats in the ESE significantly reduced Rev

dependent nuclear export activity (p < 0.01) (Fig. 6B). The greatest reduction in activity was

seen in constructs containing mutations of all seven GAA repeats (mutAll) or the three

repeats in the A purine stretch (mutA). The reduction in activity in mutA indicates this

region, while not necessary for GST-Rev binding, is required for RNA nuclear export.

Mutations in the B region (mutB, B12, B34) also significantly reduced activity. Therefore,

we conclude that the B purine stretch functions in GST-Rev binding, exon 3 inclusion, and

Rev-dependent nuclear export. Together, these results indicate the ESE sequence acts as an

RRE and that Rev mediates alternative splicing by binding at or near the ESE to disrupt

SF2/ASF interactions.

SR proteins inhibit Rev-dependent nuclear export. The finding that similar ex­

acting sequences mediate nuclear export, RNA binding, and exon inclusion suggested that

EIAV Rev directly competes with SF2/ASF for binding at a similar site on the viral pre-

mRNÀ. If so, increasing concentrations of SF2/ASF would inhibit Rev-dependent nuclear

export activity. To test this, initial studies were done to determine the linear range of Rev-

123

dependent nuclear export activity. Based on these results, Cf2th cells were transfected with

0.5 ng pRevWT and ten-fold increasing concentrations of pSF2/ASF. Results indicated a

significant, dose-dependent decrease in CAT levels in the presence of pSF2/ASF (Fig. 7).

This suggests that SF2/ASF and Rev are mutually competitive and is consistent with the

conclusion that both proteins bind to nearly identical sequences on the viral pre-mRNA.

Western blot analyses confirmed that SF2/ASF protein levels increased concomitant with

increased amounts of transfected plasmid DNA (data not shown). The level of Rev produced

by transfected Rev cDNA was below the limits of detection by Western blot. Thus, we could

not eliminate the possiblity that the decrease in Rev-mediated nuclear export activity resulted

from quantitative changes in Rev levels rather than changes in Rev binding.

Discussion

In addition to its role in nuclear export of incompletely spliced viral mRNAs, EIAV

Rev mediates alternative splicing of the four-exon multiply spliced EIAV mRNA (22).

Derse and co-workers (17) demonstrated that both GST-Rev and the SR protein SF2/ASF

cross-link in vitro with exon 3, and proposed a model wherein Rev disrupts SR protein

interactions required for exon inclusion. Consistent with this previous data, we show the

purine rich sequence is required for GST-Rev binding and for exon 3 recognition in splicing

reactions in vitro. In addition, a 57 nt sequence containing the ESE was shown to act as a

functional RRE in a heterologous nuclear export assay system. Mutation of GAA nucleotide

repeats in the ESE reduced GST-Rev binding, exon 3 splicing in vitro, and nuclear export of

ESE containing pre-mRNA. Trans-complementation assays demonstrated that SF2/ASF

inhibited Rev-dependent nuclear export in a dose-dependent manner. Therefore, both

124

SF2/ASF-mediated exon 3 splicing and Rev-mediated RNA export have similar c/s-acting

RNA requirements, and EIAV Rev and SF2/ASF appear to be mutually competitive. From

these data, we conclude that the purine rich sequence within exon 3 of EIAV is both an ESE

and a functional RRE. Extending Derse's model, we propose that Rev-mediated nuclear

export requires binding at or near the ESE, and that this results in skipping of exon 3 through

direct inhibition of SF2/ASF- ESE interactions required for recognition of exon 3 by the host

cell splicing machinery. The use of an ESE as an RRE is unprecedented among complex

retroviruses.

It is interesting that mutation of the 5' purine stretch (mutA) decreased nuclear export

but appeared to have little effect on GST-Rev binding in vitro. Studies with HIV-1 Rev also

indicate that sequences in the HIV-1 RRE are required for nuclear export but not RNA

binding (25, 26). While these observations have not been fully explained, it is likely that

RNA secondary structure may play a role. Secondary structure is a key determinant for HIV-

1 binding, multimerization, and function (7, 8,10,11, 24, 25, 27). No biochemical data is

available to date to confirm the proposed structure (17) of the EIAV ESE/RRE, and it is not

possible to predict the structural effects of the mutations used in our study. Our data would

suggest the mutation of the 5' purine stretch does not affect the primary binding site of Rev,

but may alter distant structures required for Rev-mediated nuclear export. It has been

demonstrated that HIV-1 Rev multimerization occurs only after binding to a primary site on

the RNA and furthermore, other regions of the RNA are important for secondary binding (8,

10, 32). However, we were unable to observe Rev multimerization in our RNA binding

assays, including those assays containing the ESE. Therefore, it remains unclear why mutA

exhibited reduced activity with no apparent defect in RNA binding. It is also possible that

binding of host cell proteins to the RRE may be required to facilitate Rev export activity.

Further studies will be necessary to delineate the role of this purine region in Rev-mediated

nuclear export.

Interactions of Rev-like proteins with SR proteins have been demonstrated in other

complex retroviruses. SR proteins have been shown to bind the HIV-1 RRE in a Rev-

dependent manner (28). The same study also demonstrated that excess exogenous SF2/ASF

could produce a dose-dependent inhibition of HIV-1 Rev function in vivo. We have

previously reported an inhibition of EIAV replication in activated macrophages associated

with a delay in the appearance of incompletely spliced RNAs (31). The data presented here

would suggest this inhibition may be a result of competition of SF2/ASF with Rev for

binding at the ESE. This hypothesis is supported by our data showing that excess SF2/ASF

provided in trans can inhibit Rev function in transient transfection assays. Together, these

results suggest the inhibition in activated macrophages may be due to an increase in the level

of SR proteins. It is known that expression of SR proteins varies in cells at different states of

activation and differentiation (16, 30, 33), including an increased expression of the SR

protein SRp30c in activated T-cells (30). However, little is yet known about the phenotype of

SR proteins in monocyte cells. Also, our data cannot rule out the possibility that in addition

to competing for binding at the RRE, Rev may also inhibit function via protein-protein

interactions.

Previous reports have demonstrated that mutations in the NES do not affect the

alternative splicing activity of EIAV Rev (4,18). To date, no laboratory has identified a Rev

protein which is competent in nuclear export, but deficient in alternative splicing. Therefore,

it is not clear whether alternative splicing of exon 3 is merely a consequence of Rev-

mediated nuclear export, or if it plays a separate role in virus replication. The RREs of most

complex retroviruses are located near the SU-TM cleavage site or in the 3' end of env. The

location of EIAV RRE in the 5' env is unique, and may be explained by the requirement of

the ESE for exon 3 inclusion. The env mRNA is spliced using the exon 2 splice acceptor

(Fig. 1). A singly spliced mRNA using the exon 3 splice acceptor has not been observed in

infected cells, and would encode a truncated Env protein lacking the signal peptide. A singly

spliced mRNA using the exon 4 splice acceptor is observed, which produces a truncated

transmembrane protein from the alternate start codon present in exon 1. Therefore, the use of

an ESE as an RRE may function to silence recognition of exon 3 to eliminate another singly

spliced transcript. Although a number of retroviruses utilize cis-acting sequences such as

ESEs to take advantage of cellular mechanisms of alternative splicing, EIAV appears to be

the only retrovirus to encode a trans-acting protein that directly modulates SR-mediated

alternative splicing. EIAV was the first described lentivirus and is smaller and genetically

less complex than the other lentiviruses. It is possible that the EIAV Rev-ESE interaction

may represent an transitional step in the evolution of the Rev-Rex pathway utilized by most

complex retroviruses. Interestingly, previous work in our laboratory and others suggested

that EIAV may possess two separate RREs (4, 22). In the current study, reporter constructs

containing the 57 nt ESE region showed significantly reduced activity as compared to the

ERRE-A11 reporter construct containing a larger portion of the env gene (Fig. 6A). However,

a second RRE element has not been identified, nor is it clear that such an element can

function independently of the ESE to mediate export of viral pre-mRNAs. Additional studies

will help to fully understand the biological and evolutionary significance of the EIAV Rev-

mediated export pathway.

127

Acknowledgements

We thank Yvonne Wannemuehler for technical assistance, Tom Hope for plasmids

pERRE-All, pERRE-1, and pDM138, and Sean Murphy and Prasith Baccam for statistical

analysis.

This work was supported by funds from the Carver Grant Trust (S.C.), an Iowa State

University - University of Iowa Interinstitutional grant in Biomedical Sciences (S.C. and

C.M.S.), USDA grant 96-358204-3847 (S.C.), and PHS grant AI36073 from the National

Institute of Allergy and Infectious Disease (C.M.S.).

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Figure Legends

Figure 1. Organization and splicing patterns of EIAV. Schematic of EIAV genome with

ORFs. The tat ORFs are indicated with a 't\ the first exon of rev with a 'r' (location in

genome is indicated by the shaded region). Splicing patterns and genes expressed are

indicated. The ttm ORF encodes a truncated transmembrane protein of unknown function (3).

Figure 2. Exon 3 splicing requires the purine rich sequence. (A) Diagram of RNA substrates

used for in vitro splicing showing the location of exons 2 and 3. All substrates contain the

exon 2 splice donor and exon 3 splice acceptor. Approximate location of purine rich

sequence is highlighted. (B) After incubation for 2 hr in HeLa cell splicing extracts, RNA

products were electrophoresed through 4% polyacrylamide gels and visualized by

autoradiography. Location of spliced and unspliced products is shown. The fastest migrating

products in lanes 3-5 are intron products resulting from splicing.

Figure 3. Rev inhibits exon 3 splicing. GST-Rev or GST was added at the indicated

concentrations to the splicing reactions. Location of the splicing products is indicated.

Figure 4. GST-Rev binds the ESE in exon 3. (A) Location of RNA probes, relative to exon

3, used in RNA gel mobility shift assays. The purine rich ESE sequence is highlighted. (B)

After incubation with GST or GST-Rev, radiolabeled RNAs were electrophoresed through

8% native polyacrylamide gels. Location of GST-Rev-RNA complexes is indicated with an

arrow. (C) Competition assays were performed with either 0.5 or 1 jug of the indicated excess

cold competitor RNAs. Competitors were mixed with GST-Rev 10 min. prior to the addition

of radiolabeled probe.

134

Figure 5. In vitro splicing and RNA binding of ESE mutants. (A) Sequence of two purine

stretches (designated A and B) in exon 3. GAA repeats were mutated to GCA in largest

splicing construct (Fig. 2A) and RNA probe RREp4 (Fig. 4A). (B) In vitro splicing analysis

of mutant ESE constructs. Location of splicing products is indicated. (C) RNA gel mobility

shift assays detecting GST-Rev binding to the mutant probes.

Figure 6. EIAV ESE can function as an RRE. (A) pDM138-derived reporter vectors

containing various regions of the EIAV genome. Transient transfections and CAT assays

were performed in 293 cells as described in Materials and Methods. The results are presented

as the percentage acetylation. Experiments were performed in triplicate and the results

represents at least nine independent transfections. Error bars denote the standard error of the

mean. (B) ESE mutations indicated in figure 5A were also introduced in the ERRE-1 A

reporter vector and assayed for CAT activity in the presence or absence of Rev as described

above.

Figure 7. SF2/ASF inhibits Rev-dependent nuclear export. pERRE-1 reporter plasmid was

co-transfected with 0.5 ng pRevWT and increasing amounts of pSF2/ASF. CAT levels were

quantified by ELIS A and are reported as pg CAT per normalized lysate. Results represent

the mean of six independent transfections, and the error bars denote the standard error of the

mean. Asteriks indicate values significantly different (p < 0.05) from control transfections

which contained no pSF2/ASF.

135

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141

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