Retrovirus(Raymond B. Birge, PhD)

October 9th, 2008

Viruses, Cells, andDiseases

(CIOC5125Q)

Oncogenic VirusesDefinition: A virus that is able to take residence in a cell and alter cellular growth and give properties of neoplasia. These cells are referred to as “transformed”

cells

DNA Tumor Viruses RNA Tumor Viruses

DNA Viral Genome

Viral mRNA

Viral Protein

DNA-dependent DNA polymerase

(Viral or Host)

RNA polymerase

(Host)

Examples:

Papovaviruses (Papillomavirus, SV40)

Adenoviridae (Adenovirus)

Herpesviridae (Epstein-Barr virus)

RNA Viral Genome

Viral DNA Genome (integrated)

Viral RNA Genome

Viral Protein

Reverse transcriptase

(Viral)

DNA-dependent RNA polymerase

(Host)

RNA Splicing

(Host)

Examples:

Oncovirinae (RSV, MuLV, HTLV-1,2)

Lentivirinae (HIV)

TERMINOLOGY

Oncogene: A viral or cellular gene directly responsiblefor the induction of abnormal cell proliferation.

Proto-Oncogene: A cellular gene that has the potential tobe an oncogene if its encounters mutation/de-regulation.

Tumor Suppressor Gene: A cellular gene involve in growth suppression. These genes are inactivated by deletion, mutation, of interaction with viral proteins(for example, HPV gene products).

Oncogene Hypothesis: Malignant carcinoma’s arisefrom de-regulation or mutation of cellular genes.

Basic Structure of Retrovirus; Genome encodes three basic genes(Gag, Pol, and Env)

ALV (ASLV) Avian (Sarcoma) and Leukosis Virus

Coat proteins (surface glycoantigens); Encoded by Env

Outer lipid Envelope (Derived from the host membrane)

Can be trans-membrane or cleaved. Env variationsdetermine subgroups (A-E, and J).

Capsid core proteins (core shell, includes the Matrix (MA)Capsid (CA-most abundant), and Nucleocapsid (NC)

RNA genome (2 molecules)

Polymerase (several genes; protease RT, RNase H, Integrase)

RNA genome; both RNA molecules are single stranded (+) sense and5’ cap and 3’ poly A tail. They also have a small molecule of tRNA (usually

for Trp or Lys!)

Basic Structure of Retrovirus; Genome encodes three basic genes(Gag, Pol, and Env)

ALV (ASLV) Avian (Sarcoma) and Leukosis Virus

Coat proteins (surface glycoantigens); Encoded by Env

Outer lipid Envelope (Derived from the host membrane)

Can be trans-membrane or cleaved. Env variationsdetermine subgroups (A-E, and J).

Capsid core proteins (core shell, includes the Matrix (MA)Capsid (CA-most abundant), and Nucleocapsid (NC)

RNA genome (2 molecules)

Polymerase (several genes; protease RT, RNase H, Integrase)

Coat proteins also determine trophism

Coat proteins determine trophism;Ecotrophic=infects mouseXenotrophic=infects non mouse (rat, hamster)Amphotrophic=mouse and non-mouse (human)

Topology and classification of retrovirus

A-type: Non-enveloped particles, only seen inside cells(maybe they are partially expressed endogenous virus)

B-type: Enveloped particles, with condensed core and prominent envelope spikes (MMTV)

C-type: Enveloped particles, with condensed core and few envelope spikes (ALV, RSV, HIV, HTLV)

D-type: Enveloped particles, less condensed core, fewenvelope spikes (able to super-infect C-type virus)

Taxonomy of RNA Reverse Transcribing Viruses(Family=Retroviridae)

Alpharetrovirus ALV, RSV, CT10, Y73 Sarcoma Virus Vertebrate

Betaretrovirus MMTV, Squirrel monkey retrovirus Vertebrate

Gammaretrovirus FLV, Harvey MSV, Moloney MSV Vertebrates

Deltaretrovirus BLV, Primate T-lymphocytic retrovirus Vertebrate

Lentivirus HIV-1, HIV-2, SIV, HTLV Vertebrates

Spumavirus Chimp foamy virus Vertebrates

Genus Type Hosts

Classification taxonomy depend on various factors that include genome size, assembly mechanisms, subtype, malignancies, immunodeficiency, and homology.

The basic retrovirus lifecycle

The retrovirus lifecycle in more detail

1. Binding to a receptor (fusion/internalization)Note, there are four subtypes(A-type, B-type, C-type, D-type)

2. RNA (plus strand) is copied to DNA(minus)strand. ssDNA copies to dsDNA

3. DNA, called provirus) is integrated intohost chromosome randomly

4. Full-length genomic RNA is copied from integrated DNA by pol II.

5. RNA is spliced and translated into protein

6. Virus particles assemble and bud from plasma membrane

The lifecycle in more detail

1. Binding to a receptor (fusion/internalization)Note, there are several subtypes(A-type, B-type, C-type, D-type etc)

2. RNA (plus strand) is copied to DNA(minus)strand. ssDNA copies to dsDNA

3. DNA, called provirus) is integrated intohost chromosome randomly

4. Full-length genomic RNA is copied from integrated DNA by Pol II.

5. RNA is spliced and translated into protein

6. Virus particles assemble and bud from plasma membrane

Retrovirus lifecycle is somewhatuneventful, consuming about 1%of the total cellular energy.

Retrovirus replication and integration:

5’Repeat

3’Repeat

5 ‘Unique

3’Unique

Primer binding

site

PolypurineTract

Single stranded RNA

Major elements of the retrovirus sense strand.

Direct repeats at both ends of the genome ‘terminally redundant”Primer binding site, uses a specific tRNA (15-20 nucleotides C’ to 3’ end)Polypurine Tract; Short stretch of A and G residues for initiating (+) strand

synthesisUnique 3’ region responsible which forms the promoter for the viral mRNA

5 Cap (AAAA)n

RT

Retrovirus replication:

5’Repeat

3’Repeat

5 ‘Unique

3’Unique

Primer Binding Site

Single stranded RNA

RT

RT

RT

RT

tRNA primer binds to PBS; RT extends in 3’ direction

(only virus that uses tRNA for replication!)

RNase H degrades ds RNA

(part of RT enzyme; degrades RNA/DNA duplex to ssDNA

SS DNA jumps to opposite

complementary Strand

tRNA primer binds to PBS; RT extends in 3’ direction

Polypurine Tract

Note, this sense strand does

not serve directly as mRNA

Retrovirus replication :

RTtRNA primer binds to PBS; RT

extends in 3’ direction

RNase H degrades all ds RNA except for the polypurine tract

RT

PolypurineTract

RT now extends in the sense direction

RT

RTRNA

RNase H removes remaining RNA

C’ sequences allow for

circularization

After circularization, RT acts as a DNA-dependent DNA polymerizationto make the ds DNA pro-virus

David Baltimore(MIT)

Howard Temin(U Wisconsin)

RSV virus particles contain an endogenous DNA polymerase activity

(incorporates deoxyribonucleside monophosphates into DNA and requires allfour deoxyribonucleotide triphosphates, a divalent cation,and is inactivated by RNAase)

1975 Nobel Prize in Physiology and Medicine

The next step in virus life is the DNA provirus integration, catalyzed by the integrase function of RT.

Integrase Function is part of RT, proviral insertion

Can occur with either linear of circular form of the provirus

The ends of the LTR’s have inverted repeats that are cleaved to form a staggered cut.

5’ LTR 3’ LTR

IN

IN also makes a cut in the host cell DNA, allowing permanent insertion of the entire ds provirus

Host DNA Host DNAViral DNA

DNA provirus

host hostLTR LTRgag pol env

5’ 3’gag pol env

Transcription (through cellular transcriptionalmachinery)

gag-pol polyprotein

gag polyprotein

Virion structuralproteins

Integrase; RT;RHase H, protease

Envelope glycoproteins(SU and TM)

env

5’ 3’envSubgenomic env mRNA

“A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to wide-spread metastasis. In a careful study of the growth,tests have been made to determine whether it can transmitted by a filtrate free of the tumor cells…small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl” (Rous, Nature , 1911). The cancer was named Rous' sarcoma, and Rous won the Nobel Prize in 1966 for his achievement.

A cancer-causing virus in chicken

RSV-Malignant sarcoma’s within 2 weeks

ALV-Weakly transforming virus (tumors in 2-3 months)

Growth characteristic Normal cells Tumor cells

Density dependent inhibition of growth Present Absent Growth factor requirement High LowAnchorage dependence Present AbsentProliferative life span Finite IndefiniteContact inhibition of motility Present AbsentMorphology Flat RoundedColonies in agar NO YES

Normal cells RSV transformed cells

ALV and RSV could be propagated and isolated cultured cells

How can ALV, with a relatively uneventful infectionlifecycle, induce cell transformation and neoplasia?

The DNA Provirus Hypothesis (1965-74)

RNARSV DNARSV

Infecting virus

RNARSV

Provirus Progeny Virus

The Oncogene Hypothesis(The protovirus hypothesis for

origin of cancer genes�)

DNA Altered DNADNARSV

Provirus

Hidesaburo Hanafusa:Defectiveness of Rous sarcoma virus; Virtually all acutely transforming retroviruses of animals are mixtures of replication competent helper virus and replication defective transforming virus.Replication function is provided bythe helper virus in trans

Hybridization experiments. PNAS (1970). “These results indicate that both cell types (chicken cells that contain RAV-60 in a replicating form or do not appear to contain a replicating form) contain DNA that is complementary to RNA from the avian tumor virus”.

(~1971)

ALV RSV

Weakly transforming virus (3-6 months)Genome size ~8.5 kbReplication competent

Acutely transforming virus (1-2 weeks)Genome size ~10 kbReplication deficient

What is the difference between weak and acutely transforming viruses?

Origin of Retroviral Transforming Genes

RSV (gag pol env src)r.t.

gag, pol etcsrc

cDNA

DENATURE

AL V (gag,pol,env)

isolate genomic RNA

gag, pol, env

HYBRIDIZE

unhybridizedsequences

hybridizedsequences

gag, pol, envsrc

Stehelin, Bishop and Varmus

src

Origin of Retroviral Transforming Genes

Specific src probe

HYBRIDIZE

RSV-InfectedCEF (+ control)

“Normal”chick DNA

MOUSEDROSOPHILA

HUMAN

+ + + + +

Stehelin, Bishop and Varmus

Thus: a proto-oncogene is the NORMAL progenitor gene of a viral oncogene

J.Michael Biship Harold Varmus

1989 Nobel Prize in Physiology and Medicine

Retrovirus and the Cancer Connection

ALV RSV

Weakly transforming virus (3-6 months)Genome size ~8.5 kbReplication competent

Acutely transforming virus (1-2 weeks)Genome size ~10 kbReplication deficient

Whats the difference between weak and acutely transforming viruses?

gag envpolΔenv

gag pol env src

LTR LTRgag pol env

LTR LTRgag pol

env src

8.5 kb

10 kb

ALV DNA

RSV DNA

pol

env src

gag

gag & pol proteins

Env proteins

Src proteins*

Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

*Overexpression of mutated Src tyrosine kinase leads to cell transformation

Avian Leukosis Virus Leukemogenesisby Promoter Insertional mutagenesis

ALV

ALV Integration Site5’ or 3’ of gene

c-myc gene

transcription of myc from LTR

ABERRANT REGULATION OFmyc TRANSCRIPTIONMMTV ‐int‐1

int‐2

etc.

LTR LTRgag pol env

LTR LTRgag pol

env src

8.5 kb

10 kb

ALV DNA

RSV DNA

pol

env src

gag

gag & pol proteins

Env proteins

Src proteins*

Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

*Overexpression of mutated Src tyrosine kinase leads to cell transformation

Src and tyrosine phosphorylation (Brugge and Hunter)

RSV encodes a 60 kD protein called Src

Injected RSV-bearing tumors into rabbits--generatedanti-sera against 60 kD protein

Using a technique called Immunoprecipitation, investigators showed that this anti-sera bound a protein with an unusual kinase specificity--

Shortly thereafter, many oncogenes identified in RNA viruses encoded tyrosine kinases.

In cells transformed by many Src and Abl (and other TK oncogenes)There is dramatic increase in protein tyrosine phosphosprylations

Structure of v-Src gene

SH3 SH2 SH1 (PTK catalytic)p60src (RSV)

Structure of v-Src vs c-Src genes

SH3 SH2 SH1 (PTK catalytic)v-Src

c-SrcTyr527

Structure of v-Src vs c-Src genes

SH3 SH2 SH1 (PTK catalytic)v-Src

c-Src

Tyr527

(open-high activity)

(closed-suppressed activity)

Non-receptor tyrosine kinases are generally maintained inauto-inhibited clamped structures, but very sensitive to

mutational activation and global destabilization.

Kuriyan, Rockefeller University, UC Berkely

Abelson leukemia virus encodes a cellular oncogene called abl, anothernon-receptor tyrosine kinase (like Src)

Abelson murine leukemia virus (A-MuLV)

Derived from Moloney murine leukemia virus (Mo-MuLV)Genus= gamma-retrovirusCauses a rapidly progressive lymphosarcoma in mice

6 kB

LTR LTRgag abl

9 kB

LTR LTRgag

p120 Gag-Abl

pol env

A-MuLV

Mo-MuLV

Helper or Dependovirus

Tyrosine kinases are regulated by ‘auto-clamped’structures using their SH2 and SH3 domains

Gag

Bcr CML, ALL

c-ablc-Abl

v-ablv-Abl

v-Src and v-Crk oncogene products

SH3 SH2 SH1 (PTK catalytic)

viral gag

p60src (RSV)

p47gag-crk (CT10)

PLC-γ II catalytic-1 catalytic-2

Crk was originally identified in the retroviral genome of avian retrovirus CT10

SH2gag SH31 440208 248 340 373 424

PhosphotyrosinepYxxP

Proline-rich sequencePx(L)PxK,R

p130 Cas

Paxillinc-CblEGF-RGab1XWee1

c-AblC3GSOSDOCK180Esp15JNKPI3 Kinase

v-Crk ; CT10 regulator of kinase

p47gag-crk

97

66

45

kDa

.

116

CEF

/ v-

Crk

Blot : α- P-Tyr

CEF

31

200p130 Cas

Paxillin

Binding partners* Binding partners*

* Partial List

AFAP

cAbl/Bcr-Abl

SH2 SH3-CSH3-N Y

PP

222c-Crk II

SH2 SH3-N c-Crk I

SH2 SH3-NGag v-Crk

SH2 SH3-CSH3-N YP

P Crk-L207

Crk family of adaptor proteins

Harvey and Kirsten murine sarcoma viruses Originally discovered in 1960’s by Jennifer Harvey and Weiner Kirsten (Harvey murine sarcoma virus and the Kirsten sarcoma virus)

Both viruses encoded retroviral genomes with the h-ras or k-rascellular genes. The protein products were 21 kD GTPases and later shown to have G12V mutations.

Ras-GDP Ras-GTP

Guanine-nucleotide exchange factor

GTPase activating protein (GAP)

Guanine-nucleotide exchange factor

GTPase activating protein (GAP)

G12V RasG12V Ras98% 2% 100%0%

The Field Comes Of Age

Raf

ERK

AP-1

Activated Proto-oncogenes from DNA Transfection

RESULT: RAS “Activation”is due to a SINGLE point mutation (gly val) at codon 12

Use Aluprobe

Isolate Human DNA

RESULT: A SINGLE human gene is responsible for transformingcapability

Sequence

RESULT: The gene is the HUMANc-H-ras gene !!!

Compare sequenceto NORMAL gene

Parada and Weinberg

Activated Proto-oncogenes from DNA Transfection

Human Bladder Tumorcell line DNA

Isolate high MWDNA

Isolate DNAfragments

restrictionendonuclease

TRANSFECTION

NIH 3T3fibroblasts

TRANSFORMATION

Isolate DNA (>99% mouse +8-10 human genes)

TRANSFORMATION

Isolate HumanDNA

Parada and Weinberg

• 1984- Avian erythroblastosis virus gene(v-erbB) is shown to be a truncated Epidermal Growth Factor receptor

• 1985- Simian sarcoma virus transforming gene (v-sis) is shown to be analogous to the β chain of PDGF receptor

• 1987- v-jun Avian oncogene is shown to be analogous to AP-1

Many of the Proto-oncogenes in viruses turned out to be involved in sporadic mutations in human cancers

Retrospective view of the Importance of Retroviral Oncogenes

About 25 naturally occurring oncogenes have been identified

Oncogenes in the 21st Century• STI 571 (Gleevec, Imatininb) is the first line therapy

for CML– The target protein is Bcr-Abl, a protein tyrosine kinase (Abl

was first known from a retrovirus)– Therapy resistance stems from changes in other proto-

oncogenes such as Src– Second line and combination therapies with Src inhibitors

are becoming standard• BUT-there are failures as well as successes-EGF-R

inhibitors are of limited value, so there is still much to learn

The EGF-R-Ras-Raf-Map kinase pathway is an important targeting module for cancer therapeutics

BAY439006 (Advanced kidney cancer)Raf

MAP-kinase

Transcription of growth gene

Src/PI3-kinase

(Tyrosine kinase; Abl)

EGF-R/HER2/Neu

Ras Ras InhibitorsRTK Inhibitors(MANY; ie Gefitinib)

Gleevac-STI571

AG490

Nu2048 (Myc)

v-Erb2v-Ha-Ras

v-Raf

v-Myc

v-Src

ABT-737v-p110

Memorial symposium for the contributions of Teruko Hanafusa (1998)

Nature Reviews Molecular Cell Biology 2; 467-475 (2001); THE HUNTING OF THE SRC

Homework Assignment: Due October 16th

1. How would you determine whether the virus is a retrovirus? What assays would you do, and how would you interpret your data?

Now that you are experienced virologists (after passing “Viruses, Cells, and Diseases”!),you have been invited to work on novel oncogenic virus (called C5125Q) isolated from a mouse populationin the Outback of Australia. Injection of serum or purified virus causes a acute leukemia, specific to T and B cells.

Using both specific knowledge (and your imagination) describe a scenario by which C5125Q may cause transformation. Remember, this is a novel retrovirus and not likely to behave exactly as the viruses we studied in class!

2.

1838. Recognized as a collection of disorganized, abnormal cells (Virchow)

1907-33. Cell-free filtrates induce tumors in chickens (Rous, Shope, Fujimani)

1910-25 Chromosomal abnormalities (Tyzzer, Boveri).

1915-41 Carcinogen-induced cancers, Multiple factorsinvolved--Initiation and Promotion factors (Yamigiwa, Berenblum)

1951-53 Mouse Leukemia Virus, Polyoma Virus (Gross, Friend, Stewart)

1964. Provirus Hypothesis (Temin)

1969-76. Normal cells suppress transformation--cell hybrids (Harris,Stanbridge)

1970. Reverse Transcriptase (Baltimore, Temin)

1971. Hereditary nature of retinoblastoma (Knudsen)

1976-80. Oncogene theory, cellular origin of viral transforming genes(Varmus, Bishop, Hanafusa, Vogt).

Milestones in Cancer Biology.

Milestones in Cancer Biology-part II

1979-1982. Discovery of Ras oncogene (Weinberg, Cooper, Wigler, Barbacid)

1982-1987. Activation of many genes by virus integration, many oncogenesidentified.

1988. Isolation of Retinoblastoma gene--concept of ‘tumor suppressor genes’developed. (Weinberg)

1988-1991. Multistep carcinogenesis models (Vogelstein, Liotta)

1992-1994. Programmed cell death, Bcl-2, p53 (Korsmeyer, Levine, Horvitz)

1996. Angiogenesis Inhibitors (Folkman)

1999. STI-571 (Glivac) Bcr-Abl Inhibitor, CML (Drucker, Kuriyan)

2001-06. Era of Genomics, Proteomics, to the cancer problem (Collins, Ventor,Lander)

2007. Cancer stem cell biology and tumor microenvironment (Bissell/Weinberg)