+ All Categories
Home > Documents > Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

Date post: 03-Feb-2022
Category:
Upload: others
View: 4 times
Download: 0 times
Share this document with a friend
10
JOURNAL OF VIROLOGY, 0022-538X/98/$04.0010 Sept. 1998, p. 7191–7200 Vol. 72, No. 9 Copyright © 1998, American Society for Microbiology. All Rights Reserved. Rescue of Defective Poliovirus RNA Replication by 3AB-Containing Precursor Polyproteins JONATHAN S. TOWNER,² MELISSA M. MAZANET,‡ AND BERT L. SEMLER* Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697 Received 27 February 1998/Accepted 22 May 1998 This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) {wild type}] in trans. The replication- defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the mem- brane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor poly- peptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus rep- lication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product. Poliovirus (PV), the prototypic picornavirus, replicates its genomic RNA via membranous replication complexes within the cytoplasm of an infected cell (11, 12). These complexes appear as rosette-like structures (8) and are thought to provide an environment for increased local concentrations of poliovi- rus proteins, presumably limiting diffusion within the replica- tion complex (22). Highly specific interactions between cellular and viral proteins associated with virion RNA (vRNA) and cellular membranes result in the formation of PV replication complexes. These interactions include tight membrane-protein associations by PV proteins 3AB (17, 40, 45, 47, 52) and 2C (15, 20, 48) combined with specific RNA-protein interactions between the PV 59 noncoding region (59NCR) and the viral polypeptide 3CD (2, 3). In addition, the presence of the 59 terminal ;100 nucleotides (nt) of PV RNA mediates an in vitro interaction between 3CD and the cellular protein poly(rC) binding protein 2 (21, 36). The viral polypeptide 3AB (24, 54) and the cellular protein EF-1a (24) also appear to complex with 3CD and the 59 end of the PV genome. Interac- tions between the viral proteins 3D and 3AB have been doc- umented (25, 29, 37), as have RNA-protein interactions involv- ing the 39 ends of positive- and negative-strand picornavirus RNAs (4, 39, 50). While many of these studies have focused on individual molecular interactions, little is known about the viral polyprotein subunits necessary for the initial assembly of the vRNA replication complex. For PV, like all picornaviruses, mature gene products are specifically processed from viral precursor polyproteins (42). The efficient and highly regulated protein processing cascade produces cleavage products with functions distinct from those of their precursor proteins. Due to the short half-lives of large precursor proteins, it has been difficult to determine the composition of assembly intermedi- ates. An additional control of RNA replication relies on the trans- lation of PV RNA; that is, translation of a particular genome is a prerequisite for that genome to be competent for replica- tion due to the requirement for either a cis-acting viral pro- tein(s) or ribosomal passage over the RNA template (34). A relationship between ribosomal passage and RNA replication stems from the observation that all naturally occurring defec- tive interfering particles contain deletions in the capsid region (P1 [Fig. 1]) that maintain the reading frame (28). The require- ment for translation prior to RNA replication was later con- firmed by using genetically engineered replicons in which rep- lication occurred only if deletions were in frame and the P2-P3 region was left intact (except for the N terminus of viral pro- tein 2A) (16). The apparent cis dominance of translation over replication does not in theory preclude the utilization of some of the viral nonstructural proteins in trans. For example, pas- sage of the ribosome could transiently expose a cis-acting rep- lication determinant present on the RNA template, while the active replication proteins in the complex could consist of mixed viral proteins, some of which were synthesized from other viral mRNAs. Complementation of site-specific lesions in trans has been assayed by providing the wild-type gene product(s) through a helper virus (6, 7, 14, 18, 22, 26, 32, 49) and through novel approaches using dicistronic RNAs or amber-suppressing cell lines (13, 34). The collective results of such studies indicate that each viral protein likely has multiple functions, some of which can be complemented in trans and some of which can- not, depending on whether a mutation exerts its effect at the level of the precursor protein or at the level of the mature cleavage product. Previous experimental approaches often utilized a helper virus or helper RNA to provide the complementing gene prod- * Corresponding author. Mailing address: Department of Microbi- ology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697. Phone: (949) 824-7573. Fax: (949) 824- 8598. E-mail: [email protected]. ² Present address: Centers for Disease Control and Prevention, Spe- cial Pathogens Branch, Division of Viral and Rickettsial Diseases, Atlanta, GA 30333. ‡ Present address: Department of Molecular Biology and Biochem- istry, University of California, Irvine, CA 92697. 7191 on November 22, 2018 by guest http://jvi.asm.org/ Downloaded from
Transcript
Page 1: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Sept. 1998, p. 7191–7200 Vol. 72, No. 9

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Rescue of Defective Poliovirus RNA Replication by3AB-Containing Precursor Polyproteins

JONATHAN S. TOWNER,† MELISSA M. MAZANET,‡ AND BERT L. SEMLER*

Department of Microbiology and Molecular Genetics, College of Medicine,University of California, Irvine, California 92697

Received 27 February 1998/Accepted 22 May 1998

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirusRNA by providing a replicase/polymerase precursor polypeptide [P3(wt) {wild type}] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the mem-brane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor poly-peptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-freetranslation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement thedefective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue ofRNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymeraseor an inactive proteinase domain displayed differential abilities to trans complement the RNA replicationdefect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus rep-lication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly ratherthan as the mature viral cleavage product.

Poliovirus (PV), the prototypic picornavirus, replicates itsgenomic RNA via membranous replication complexes withinthe cytoplasm of an infected cell (11, 12). These complexesappear as rosette-like structures (8) and are thought to providean environment for increased local concentrations of poliovi-rus proteins, presumably limiting diffusion within the replica-tion complex (22). Highly specific interactions between cellularand viral proteins associated with virion RNA (vRNA) andcellular membranes result in the formation of PV replicationcomplexes. These interactions include tight membrane-proteinassociations by PV proteins 3AB (17, 40, 45, 47, 52) and 2C(15, 20, 48) combined with specific RNA-protein interactionsbetween the PV 59 noncoding region (59NCR) and the viralpolypeptide 3CD (2, 3). In addition, the presence of the 59terminal ;100 nucleotides (nt) of PV RNA mediates an invitro interaction between 3CD and the cellular proteinpoly(rC) binding protein 2 (21, 36). The viral polypeptide 3AB(24, 54) and the cellular protein EF-1a (24) also appear tocomplex with 3CD and the 59 end of the PV genome. Interac-tions between the viral proteins 3D and 3AB have been doc-umented (25, 29, 37), as have RNA-protein interactions involv-ing the 39 ends of positive- and negative-strand picornavirusRNAs (4, 39, 50). While many of these studies have focused onindividual molecular interactions, little is known about theviral polyprotein subunits necessary for the initial assembly ofthe vRNA replication complex. For PV, like all picornaviruses,mature gene products are specifically processed from viralprecursor polyproteins (42). The efficient and highly regulated

protein processing cascade produces cleavage products withfunctions distinct from those of their precursor proteins. Dueto the short half-lives of large precursor proteins, it has beendifficult to determine the composition of assembly intermedi-ates.

An additional control of RNA replication relies on the trans-lation of PV RNA; that is, translation of a particular genomeis a prerequisite for that genome to be competent for replica-tion due to the requirement for either a cis-acting viral pro-tein(s) or ribosomal passage over the RNA template (34). Arelationship between ribosomal passage and RNA replicationstems from the observation that all naturally occurring defec-tive interfering particles contain deletions in the capsid region(P1 [Fig. 1]) that maintain the reading frame (28). The require-ment for translation prior to RNA replication was later con-firmed by using genetically engineered replicons in which rep-lication occurred only if deletions were in frame and the P2-P3region was left intact (except for the N terminus of viral pro-tein 2A) (16). The apparent cis dominance of translation overreplication does not in theory preclude the utilization of someof the viral nonstructural proteins in trans. For example, pas-sage of the ribosome could transiently expose a cis-acting rep-lication determinant present on the RNA template, while theactive replication proteins in the complex could consist ofmixed viral proteins, some of which were synthesized fromother viral mRNAs.

Complementation of site-specific lesions in trans has beenassayed by providing the wild-type gene product(s) through ahelper virus (6, 7, 14, 18, 22, 26, 32, 49) and through novelapproaches using dicistronic RNAs or amber-suppressing celllines (13, 34). The collective results of such studies indicatethat each viral protein likely has multiple functions, some ofwhich can be complemented in trans and some of which can-not, depending on whether a mutation exerts its effect at thelevel of the precursor protein or at the level of the maturecleavage product.

Previous experimental approaches often utilized a helpervirus or helper RNA to provide the complementing gene prod-

* Corresponding author. Mailing address: Department of Microbi-ology and Molecular Genetics, College of Medicine, University ofCalifornia, Irvine, CA 92697. Phone: (949) 824-7573. Fax: (949) 824-8598. E-mail: [email protected].

† Present address: Centers for Disease Control and Prevention, Spe-cial Pathogens Branch, Division of Viral and Rickettsial Diseases,Atlanta, GA 30333.

‡ Present address: Department of Molecular Biology and Biochem-istry, University of California, Irvine, CA 92697.

7191

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 2: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

ucts in infected or transfected cells. vRNAs produced in cellsfrom such approaches most likely generate individual RNAreplication complexes that are physically separated from eachother in the cytoplasm. By the time viral proteins are generatedto levels sufficient for diffusion and effective complementation,polypeptides in the replication complexes may be processed orassembled such that they are no longer competent for subunitexchange. In this study, we attempted to circumvent the prob-lem of inaccessibility to the PV replication complex by using aHeLa cell-free replication assay to produce potential comple-menting viral proteins during the initial stages of complexformation. We addressed four questions. (i) What polyproteinsubunit delivers the RNA replication function of viral protein3AB during assembly of the replication complex? (ii) Can this3AB-containing assembly intermediate by provided in trans?(iii) Does this assembly subunit need to be proteolytically ac-tive? (iv) Does 3AB need to be physically linked to the activeviral RNA polymerase during complex assembly? Our resultsshow that a 3AB mutation causing a severe RNA replicationdefect can be efficiently complemented in trans by providingthe large replicase precursor (P3) but not the mature 3ABpolypeptide. Furthermore, the rescuing trans-P3 protein mustcontain an active 3C proteolytic domain but not an activepolymerase domain. Possible mechanisms for complementa-tion are discussed.

MATERIALS AND METHODS

Plasmids and cloning. Construction of plasmid pSP6-PV1 was the result of afive-fragment ligation in which pT7-PV1 (23) was first digested with StuI andBglI, yielding three fragments: 1142-nt StuI to BglI (pBR322 nt 3480), BglI(pBR322 nt 3480) to BglI (PV nt 5318), and BglI (PV nt 5318) to BglI (PV nt 35).These three fragments were then gel purified and joined by T4 DNA ligase in thepresence of two synthetic oligonucleotide cassettes (cassette 1 contained theoligonucleotide 59-CCTATTTAGGTGACACTATAGTTAAAACAGCT-39 an-nealed to the oligonucleotide 59-GTTTTAACTATAGTGTCACCTAAATAGG-39; cassette 2 contained the oligonucleotide 59-CTGGGGTTGTACCCACCCCAGAGGCCCACG-39 annealed to the oligonucleotide 59-GGGCCTCTGGGGTGGGTACAACCCCAGAGCT-39) which together contain the first 35 nt of

poliovirus type 1 (PV1) placed immediately downstream of the SP6 promotersequences such that a single guanosine residue separates the SP6 promoter andthe first nucleotide of the PV1 sequence.

To produce significant levels of either 3AB or precursor polypeptide P3 intrans, sequences encoding these proteins were cloned into a phage T7-basedtranscription plasmid immediately downstream of a modified PV1 internal ribo-some entry site (IRES) in which sequences present in the variable region (nt 641to 722) not essential for efficient translation initiation (1, 27) were removed andthe ATG at nt 743 was placed within an NcoI site to facilitate cloning. Togenerate such plasmids [pT7-59NCR-P3wt, pT7-59NCR-P3(F69H), pT7-59NCR-3AB(wt), and pT7-59NCR-3AB(F69H)], plasmid pT7-59NCR (23) was digestedwith StuI and EaeI (PV nt 627), and the ;650-nt fragment containing the T7promoter and the first 627 nt of the PV 59NCR was gel purified. In parallel,vector sequences were prepared by digesting pT7-59NCR with StuI and EcoRI.Half of the digested vector was incubated with the Klenow fragment of Esche-richia coli DNA polymerase (generating a blunt end at the EcoRI site), and theresulting 2.3-kb fragments were gel purified. The DNAs encoding both 3AB (wt[wild type]), P3(wt), and P3(F69H) sequences were amplified by PCR frompT7-PV1(wt) or pT7-PV1(F69H) DNA, using the oligonucleotide 59-AGATCCATGGGACCACTCCAGTATAAA-39 (which encodes an NcoI site surroundingthe start codon [underlined]) along with either the oligonucleotide 59-GACTGAATTCTATTGTACCTTTGCTG-39 (for 3AB), which contains an EcoRI siteimmediately following the stop codon (underlined), or the oligonucleotide 59-TGTACTCGAGGACTGAGGTAGGGTTACT-39 (for P3), which contains anXhoI site 18 nt downstream of the natural stop codon in 3Dpol (underlined). Toclone the 3AB sequences into plasmid pT7-59NCR, the 3AB-encoding PCRfragment was then digested with both EcoRI and NcoI, gel purified, and incu-bated in the presence of T4 DNA ligase along with the ;650-nt StuI-to-EaeIfragment, the ;2.3-kb StuI-to-EcoRI (not end filled) fragment, and an oligonu-cleotide cassette consisting of the oligonucleotide 59-GGCCATCCGGTGAAATCAGACAATTGTATCAC-39 annealed to the oligonucleotide 59-CATGGTGATACAATTGTCTGATTTCACCGGAT-39. The oligonucleotide cassettecontained the PV1 59NCR sequences from PV1 nt 627 to the ATG at PV1 nt 743(placed in the context of an NcoI restriction site) with PV1 nt 641 to 722removed. To clone the sequences for P3(wt) downstream of the modified PV1IRES, the P3-encoding PCR fragment was treated like that for the 3AB DNAexcept that the DNA was digested with XhoI (blunt ended with Klenow enzyme)instead of EcoRI and the ;2.3-kb vector fragment was blunt ended as well at theEcoRI site prior to ligation with T4 DNA ligase. To clone the K61L mutation in3Dpol from the pExcalibur plasmid described by Richards et al. (38), pExcaliburwas digested with BglII and PvuII, releasing a 1,452-nt fragment (correspondingto the sequences encoding the C-terminal half of 3C plus the N-terminal half of3D), which was then gel purified. This 1,452-nt fragment was incubated in thepresence of T4 DNA ligase with gel-purified pT7-59NCR-P3(wt) that had beendigested with PvuII and BglII and gel purified. The cloning of the C147A mu-

FIG. 1. Schematic representation of full-length and subgenomic PV RNAs. (A) Schematic representation of the PV genomic structure and viral protein organizationwhere the viral structural proteins are derived from the P1 portion of the genome and the nonstructural proteins are derived from the P2 and P3 portions of the genome.Contained within the PV1 59NCR are the sequences that comprise the IRES. Note that the region of the genome designated VPg is also referred to as 3B. (B)Representation of the full-length and genetically engineered subgenomic RNAs used in this study. The PV1(wt) and PV1(F69H) RNAs contain a nonviral guanosineresidue at the 59 end of SP6-transcribed RNAs. The coding sequences for any of the P3 or 3AB proteins (derived from subgenomic RNAs) are preceded immediatelyby the PV1 IRES (modified as described in Materials and Methods). An 3 denotes the approximate location of the point mutation. Note that each P3-encoding RNAcontains 18 nt of the 39NCR immediately downstream of the authentic stop codon in 3D, a result of the 39NCR primer used in the original amplification of the P3sequences prior to cloning.

7192 TOWNER ET AL. J. VIROL.

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 3: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

tation in 3C to generate pT7-59NCR-P3(C147A) was performed by digesting a3CD expression vector (35) with BglII and AccI followed by the purification ofthe 617-nt DNA fragment encoding the 3C mutation. This 617-nt fragment wasthen cloned into pT7-59NCR-P3(wt) that had been digested with AccI and BglIIand gel purified.

RNA transcriptions. Prior to transcription, the full-length pSP6-PV1 cloneswere linearized with EcoRI, while the pT759-NCR-P3 and pT759-NCR-3ABclones were linearized with AatII, followed by end-fill repair using Klenowenzyme. Individual RNA transcription reactions were performed as described byCharini et al. (14), with the modifications described in Towner et al. (52).

Coupled in vitro translation/replication assays. Assays were performed essen-tially by the method described by Todd et al. (51) in which the HeLa initiationfactor was prepared as described by Brown and Ehrenfeld (9) and the HeLa S10extract was prepared as described by Barton et al. (5). Minor differences or newmodifications in either the extract preparation or the translation/replicationassay were as follows: (i) HeLa cells were resuspended in hypotonic buffer in avolume equal to 120% (vol/vol) of that of the cell pellet volume; (ii) eachreplication reaction mixture consisted of 51% (vol/vol) HeLa S10, 18% (vol/vol)initiation factor preparation, 21% (vol/vol) diethylpyrocarbonate-treated H2O,and 10% (vol/vol) 103 mix containing (at 103) 10 mM ATP, 2.5 mM GTP, 2.5mM UTP (no CTP was added), 600 mM potassium acetate, 300 mM creatinephosphate (Boehringer Mannheim), 4 mg of creatine kinase (Boehringer Mann-heim) per ml, and 155 mM HEPES-KOH (pH 7.4) (the composition of the 103mix was previously described by Barton et al. [5]); and (iii) reaction mixtures (50ml) were programmed with in vitro-transcribed RNA at a concentrations of 7.7nM for full-length RNAs and 3.6 nM for P3 and 3AB mRNAs, unless otherwisespecified. Each 50 ml of reaction mixture was divided into portions containing 10ml and 40 ml. The 10-ml portions (containing an additional 10.5 mCi of [35S]me-thionine [Amersham] at .1,000 Ci/mmol) were used for translation analysis,while each 40-ml portion was used for analysis of RNA synthesis. All reactionswere incubated for 6 h at 30°C, at which time the translation reaction mixtureswere diluted in Laemmli sample buffer, boiled, and subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis through a 12.5% gel. The replicationreaction mixtures were subjected to centrifugation at 15,000 3 g for 15 min at 4°Cand subsequently resuspended in 5 ml of buffer (50 mM HEPES [pH 8.0], 3 mMMgCl2, 10 mM dithiothreitol, 0.5 mM each ATP, GTP, and UTP) containing 25mCi of [a-32P]CTP and incubated for an additional hour at 37°C. Total RNA wasthen extracted from each sample; the RNA was ethanol precipitated and, fol-lowing centrifugation, resuspended in diethylpyrocarbonate-treated H2O. TotalRNA was then subjected to gel electrophoresis on a 1.1% agarose Tris-borate gelcontaining ethidium bromide. In an adjacent lane, ;500 ng of vRNA was loadedas a marker to visualize the mobility of PV single-stranded RNA (ssRNA). Forexperiments in which guanidine HCl was used to inhibit RNA replication tosynchronize RNA synthesis (method in reference 5), each replication reactionmixture was resuspended in a total volume of 25 ml of replication extract mixedtogether as described above (item ii) containing 25 mCi of [a-32P]CTP.

RESULTS

Rationale for plasmid constructions and mutations. To ex-amine the mechanism of delivery of PV protein 3AB to theRNA replication complex, we focused on complementation ofa replication-defective lesion in the putative VPg precursorprotein, 3AB. The replication-defective lesion chosen for studycodes for a phenylalanine-to-histidine change at amino acid 69(F69H) in the hydrophobic domain of PV protein 3AB. TheF69H mutation was originally designed to disrupt 3AB mem-brane association by introducing a charged residue in a puta-tive amphipathic helix. The region of the hydrophobic domaincontaining the F69H lesion was later found to be nonessentialfor membrane association (52). However, yeast two-hybridstudies suggested that this lesion disrupted protein-protein in-teractions between 3AB and itself (homodimer interactions) aswell as with the polymerase 3Dpol (55), suggesting that themutation may disrupt subunit interactions during replicationcomplex formation. When full-length RNA harboring theF69H lesion (a two-nucleotide change of UUC to CAC) wastransfected into HeLa cell monolayers and incubated at either33 or 37°C, 100- to 1,000-fold more RNA was required to seedelayed but comparable levels of cytopathic effects relative tothose seen for wild-type PV1 RNA. Sequence analysis of RNAisolated from mutant virus recovered following transfections ofPV1(F69H) RNA revealed that the adjacent amino acid atposition 70 was changed to either valine or threonine and thatthe original F69H mutation in the absence of a second-site

reversion was never recovered (data not shown). This resultindicates that the F69H lesion in 3AB causes a debilitatingreplication defect that results in a quasi-infectious RNA. Fur-thermore, in vitro translation of PV1(F69H) RNA showed noobvious defects in translation efficiency or proteolytic process-ing compared to that of the wild type. Our data suggested thatthe F69H mutation in 3AB caused a severe defect in viralreplication at the level of RNA synthesis (see Fig. 3), makingthis lesion a good candidate for trans-complementation studies.

To maximize the cell-free replication efficiency of in vitro-transcribed RNA, the PV1 cDNA [including the full-lengthcDNA encoding the 3AB(F69H) mutation] was recloned (asdescribed in Materials and Methods) under the control of thebacteriophage SP6 promoter instead of the bacteriophage T7promoter. Our rationale for this recloning was that the T7RNA polymerase requires two guanosine residues downstreamof the phage T7 promoter element for efficient transcriptioninitiation, resulting in the placement of two nontemplatedguanosine residues 59 of the authentic PV sequence. The SP6RNA polymerase requires only a single guanosine residue forefficient transcription initiation leading to a more authentic invitro transcript. Side-by-side comparisons using wild-type T7-or SP6-transcribed PV1 RNA revealed that the use of SP6-derived RNA results in a 10-fold increase in in vitro productionof infectious virus in the HeLa cell-free viral replication assay(data not shown). It is noteworthy that in vitro translation/replication reactions programmed with SP6-transcribed RNAyielded approximately 1 log less infectious virus per ml ofextract (data not shown) than those programmed with wild-type purified vRNA despite displaying approximately equallevels of translation. For a comparison of translation and RNAreplication levels directly, see Fig. 3A (lanes 3 and 8) and 4B(lanes 4 and 9). The decrease in RNA replication in the SP6-PV1 programmed reaction (Fig. 3B, lane 4) relative to thatprogrammed with vRNA(wt) (Figure 3B, lane 9) is likely dueto the single nontemplated guanosine at the 59 end of thetranscript.

Finally, to gain insights into the possible mechanisms ofcomplementation, a number of mutated versions of the P3polyprotein were studied. Two mutations were used in thecomplementation analysis. (i) C147A is a mutation in whichthe active site cysteine of 3C has been mutated to an alanine,abolishing the proteinase activity of 3C (30). This 3C lesion inP3 is referred to as P3(3C*). (ii) K61L (also referred to as m61)is a mutation in 3D which renders the RNA-dependent RNApolymerase completely inactive for strand elongation (38).This 3D mutation in P3 is referred to as P3(3D*). All of theplasmid constructs and mutations are outlined in Fig. 1.

Inability of 3AB(F69H) to exhibit trans-dominant effects.We reasoned that if 3AB is a soluble component of the PVRNA replication complex, then cotranslation of a replication-defective form of 3AB might be able to inhibit the replicativeabilities of wild-type RNA replication complexes. When either3AB- or 3AB(F69H)-encoding RNA was cotranslated withPV1 vRNA(wt) (Fig. 2A, lanes 1 and 2), the overall levels oftranslation were decreased significantly but equally with re-spect to vRNA(wt) alone (Fig. 2A, lane 3). Reduced transla-tion levels may be due to the increased concentrations of PVIRESs present when the two mRNAs are mixed together,suggesting that limiting levels of translation factor(s) arepresent in the extract. For reasons that are unclear, themRNAs encoding 3AB consistently translated less efficientlythan equimolar amounts of either PV1 RNA or P3-encodingRNA (data not shown). In the corresponding replication as-says, no differences in the levels of 32P-labeled single-strandedor replicative intermediate/replicative form (RI/RF) vRNA

VOL. 72, 1998 trans COMPLEMENTATION BY A POLIOVIRUS POLYPROTEIN 7193

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 4: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

were observed when PV1 vRNA(wt) was incubated in thepresence of either 3AB(wt) or 3AB(F69H) mRNA (Fig. 2B,lanes 3 and 4).

Based on the experiments performed by Lundquist andMaizel (33), which showed that the functional RNA replicationcomplex forms very early during the PV replication cycle, it ispossible that there is a only a small kinetic window in which3AB is able to undergo subunit exchange with a competentreplication complex. To maximize the opportunity for 3ABexchange, we presynthesized either 3AB(wt) or 3AB(F69H)for 30 min prior to the addition of vRNA. The result of thisexperiment is shown in Fig. 2A, lanes 4 to 8; lanes 7 and 8 showthe levels of 3AB generated during the 30-min preincubationperiod. The F69H mutation confers an obvious electrophoreticmobility difference from that of 3AB(wt). The translation andreplication assays are shown in lanes 4 and 5 of Fig. 2A andlanes 6 and 7 of Fig. 2B, respectively. Equivalent RNA repli-cation levels in the presence of 3AB(wt) or 3AB(F69H), eitherpresynthesized or cotranslationally synthesized, demonstratethat 3AB(F69H) was not able to exert a trans-dominant effect.

3AB(wt) is unable to rescue the replication defect of PV1(F69H). Since we were unable to detect the ability of 3AB(F69H) to effect a decrease in wild-type RNA replication, weattempted to measure 3AB complementation by assaying for3AB(wt) trans rescue of the defective replication phenotype ofthe full-length PV1 transcript encoding the 3AB(F69H) muta-tion. The results of such an experiment in which either3AB(wt) or 3AB(F69H) was cotranslated with PV1(wt) orPV1(F69H) RNA are shown in Fig. 3. The levels of [35S]me-thionine-labeled viral proteins synthesized from either of thefull-length PV1 transcripts were equivalently decreased whencotranslated with either of the 3AB mRNAs (Fig. 3A; comparelanes 1 and 2 to lane 3 and compare lanes 4 and 5 to lane 6);significant levels of 3AB proteins were produced in all of thetranslation reactions. Figure 3B shows the levels of 32P-labeledvirus-specific RNA synthesized in parallel replication reactionsto those shown in panel A. Comparison of lane 5 to lane 6

reveals no detectable virus-specific RNA synthesis, indicatingthat the presence of 3AB(wt) in trans was unable to rescue thereplication defect in 3A, a result documented even when longexposures of the autoradiographs were examined (data notshown). This result is consistent with the inability of 3AB(F69H) to exhibit a trans-dominant effect on PV1(wt) RNAreplication. As seen in each RNA replication assay and themock-treated reaction (Fig. 3B, lane 8), the abundant 28S and18S rRNAs (migrating below the ssRNA species in Fig. 3B) are32P labeled, the likely result of an endogenous terminal trans-ferase activity present in the HeLa cell extracts. Our datasuggested that the F69H lesion may exert its effects at the levelof RNA secondary structure or that protein 3AB may be un-able to enter the replication complex unless provided as part ofa larger precursor protein that is a normal component of com-plex assembly.

Complementation by P3(wt) but not 3AB(wt). To test thehypothesis that the F69H lesion in 3AB exerts its effects in aprecursor polyprotein larger than 3AB, we tested the ability ofthe 3AB-containing precursor protein, P3, to trans comple-ment the replication defect of PV1(F69H). Figure 4A (lanes 3to 8) shows that the levels of [35S]methionine-labeled proteinssynthesized from the full-length RNAs are all approximatelyequal (for reference, compare levels of protein 2A) whentranslated alone or in the presence of 3AB- or P3-encodingmRNA. Translations programmed with equimolar amounts ofRNA for either 3AB or P3 in the absence of any full-lengthPV1 mRNA are shown in Fig. 4A, lanes 9 and 10. Productionof 3AB (lane 9) can be seen in the long exposure in Fig. 4A. Itis important to note that the P3 polyprotein is proteolyticallyactive when translated alone (lane 10) or when cotranslatedwith PV1 mRNA (lanes 5 and 8), generating the cleavageproducts 3BCD, 3CD, 3AB, and 3A.

Parallel 32P-labeled RNA synthesis reactions are shown inFig. 4B. As shown in this experiment, no inhibition or enhance-ment of RNA synthesis is seen when PV1(wt) mRNA is co-translated with P3(wt) mRNA, and only a slight inhibition is

FIG. 2. Inability of 3AB(F69H) to exhibit trans-dominant effects. (A) [35S]methionine-labeled in vitro translations that were programmed either singly or doublywith 7.7 nM vRNA(wt), 36 nM 3AB(wt), or 36 nM 3AB(F69H) RNA. The sample shown in lane 9 shows the mock reaction that was not programmed with any RNA;lanes 7 and 8 show the translation of each of the 3AB RNAs during the 30-min preincubation. Note that the F69H mutation in 3A causes a distinct electrophoreticmobility difference which allows for the identification of 3AB(wt), if present, in the same reaction. Lanes 1 to 3 show reactions in which the mRNAs were cotranslated,while those in lanes 4 to 6 contained the presynthesized 3AB proteins. (B) The corresponding 23P-labeled RNA replication reactions that were performed in parallelwith the [35S]methionine-labeled translations. Lane 2 was not programmed with any RNA. Lanes 3 to 5 show reactions in which the mRNAs were cotranslated, whilethose in lanes 6 to 8 contained the presynthesized 3AB proteins. Positions of RI/RF RNA and ssRNA are shown on the right. (C) Ethidium bromide staining of theagarose gel following electrophoresis of the replication reactions to demonstrate even loading of total RNA in each lane. The photograph was taken just before theagarose gel was dried and exposed to film. The mobilities of both 28S and 18S rRNAs are indicated along with the 7.5-kb vRNA marker (lane 1) used to show themobility of full-length PV ssRNA.

7194 TOWNER ET AL. J. VIROL.

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 5: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

seen when wild-type PV1 is cotranslated with 3AB(wt) (com-pare lane 4 to lanes 6 and 5). However, when PV1(F69H)mRNA is cotranslated with P3(wt) mRNA, a six- to sevenfoldenhancement of ssRNA accumulation (quantitated by Phos-

phorImager [Molecular Dynamics] analysis) can be seen overthat for PV1(F69H) alone (compare lane 7 to lane 9), dem-onstrating that P3(wt) is able to partially rescue the replicationdefect. In contrast, 3AB(wt) causes inhibition of RNA repli-

FIG. 3. Inability of 3AB(wt) to rescue PV1(F69H). The overall format is like that described in the legend to Fig. 2 except that the RNAs used to program eachparallel translation and replication reaction are PV1(wt), PV1(F69H), 3AB(wt) (36 nM), and 3AB(F69H) (36 nM). The mock controls are shown in lanes 7 and 8 ofpanels A and B, respectively, while reactions programmed with vRNA(wt) are shown in lanes 8 and 9 of panels A and B, respectively.

FIG. 4. Efficient complementation by P3(wt) but not 3AB(wt). The overall format is like that described in the legend to Fig. 2 except that the RNAs used to programeach parallel translation and replication reaction are PV1(wt), PV1(F69H), 3AB(wt), and P3(wt). The mock controls are shown in lanes 2 and 3 of panels A and B,respectively. A long exposure of the polyacrylamide gel is shown in the bottom portion of panel A to show the production of 3AB(wt) in lane 9. Lane 10 of panel Ashows the translation of P3(wt) in the absence of any full-length PV1 RNA. In addition to the mock controls shown, PV1(wt) was translated and replicated in thepresence of 2 mM guanidine HCl (A, lane 1; B, lane 2) to demonstrate that the radiolabeled bands marked “ss vRNA” and “RI/RF” are indeed specific products ofPV RNA replication. The asterisks denote reactions carried out in the presence of 2 mM guanidine HCl.

VOL. 72, 1998 trans COMPLEMENTATION BY A POLIOVIRUS POLYPROTEIN 7195

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 6: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

cation of PV1(F69H), which agrees with what was observed inthe experiment shown in Fig. 3B. The reason for the generalinhibition of RNA replication in the presence of 3AB, despiteequal levels of translation, is not fully understood but has beenobserved at multiple 3AB mRNA concentrations (data notshown). As an additional control to demonstrate that the 32P-labeled RNAs were indeed authentic products of in vitro PVRNA replication, we carried out a parallel reaction using PV1(wt) vRNA in which 2 mM guanidine HCl was added (lane 1 inFig. 4A and lane 2 in Fig. 4B). This concentration of guanidineHCl is known to specifically inhibit PV RNA replication (10)without affecting translation.

Dose-dependent rescue by P3(wt). Based on the ability of P3to trans rescue a 3AB replication defect (Fig. 4), it was hypoth-esized that if intact or proteolytically processed P3 was ableto undergo successful subunit exchange with the replicationcomplex, then such rescue effects should be dose dependent.Therefore, increasing amounts of P3(wt) mRNAs were co-translated with a constant amount of either PV1(wt) or PV1(F69H) mRNA (Fig. 5). The quantities of P3 mRNAs rangedfrom 0.9 to 7.2 nM, based on our unpublished observationsthat PV1 IRES concentrations of 10 to 15 nM decrease theoverall levels of translation. As mentioned above, this may bedue to the limiting levels of translation factors present in thecell extract. As shown in Fig. 5A, the levels of PV proteinsgenerated from full-length PV1 RNAs were approximatelyequal over the entire range of cotranslated P3 mRNAs tested(lanes 3 to 10). However, the level of P3-specific products didincrease with increasing amounts of P3 mRNA (compare 3CD,3BCD, and P3 levels in lanes 3 to 6 and 7 to 10 of Fig. 5A). Theresults of the parallel RNA replication reactions (Fig. 5B)demonstrate a dose-dependent rescue of the PV1(F69H) RNAreplication defect by P3(wt) proteins (lanes 8 to 11). A maxi-mum level of ;5-fold rescue of ssRNA was observed at 3.6 nMP3 mRNA (lane 10), a result consistent with that shown in Fig.4 in which the same amount of P3 mRNA was used. Theseresults indicate not only that P3 is able to complement thereplication defect in trans but also that the primary defect ofthe F69H lesion in 3AB is at the protein level and not at thelevel of RNA secondary structure in the mutated genome.

trans dominant inhibition of RNA replication by P3(F69H).If P3(wt) is able to trans rescue a replication-defective com-plex, then a logical prediction is that a mutated version of P3will exert a trans-dominant effect on a wild-type RNA replica-tion complex. To test this hypothesis, increasing amounts ofeither P3(wt)- or P3(F69H)-encoding mRNA were added toreplication reactions programmed with a constant amount ofPV1(wt) mRNA. The concentrations of P3 RNAs (1.8 to 7.2nM) were similar to those chosen for the previous dose-depen-dent rescue experiment (Fig. 5). Since these cell-free replica-tion reactions are capable of translating genomic PV RNA thatis synthesized during the in vitro reaction (53), the primary 6-hincubations were carried out in the presence of 2 mM guani-dine HCl to inhibit RNA replication. This guanidine reversalmethod (5) thus eliminates any amplification effects that mightresult from a trans-dominant phenotype of P3(F69H). It alsogreatly diminishes the possibility of RNA recombination whichhas recently been shown to take place in these cell extracts (19,46).

The results of the trans-poisoning experiment are shown inFig. 6. The data displayed in Fig. 6A show that the overalllevels of translation from the PV1(wt) mRNAs are similarthroughout the range of P3 mRNA concentrations tested(lanes 3 to 9). In Fig. 6B, however, the results indicate thatincreasing amounts of the P3(wt) mRNA have no effect on theoverall levels of PV RNA replication (lanes 5 to 7), while adose-dependent inhibition of RNA replication can be seenwith increasing amounts of P3(F69H) mRNA (lanes 8 to 10).The dose-dependent poisoning of RNA synthesis by P3(F69H)ranges from 2.7-fold at 1.8 nM to 19-fold at 7.2 nM. The19-fold decrease could be a slight overestimate due to the smalldecrease in translation seen in lanes 8 and 9 of Fig. 6A. Theseresults are consistent with those shown in Fig. 4 and 5, and,taken together, indicate that P3 is likely to be a major subunitin the initial assembly of the RNA replication complex. Giventhe sensitivity of PV1(wt) replication to trans poisoning byP3(F69H) but not by 3AB(F69H), these data further indicatethat the exchangeable 3AB-containing subunit is P3 and not3AB. In addition, the data demonstrate that P3(wt) protein is

FIG. 5. Dose-dependent rescue of PV1(F69H) by P3(wt). The overall format is like that described in the legend to Fig. 2 except that the RNAs used to programeach parallel translation and replication reaction are PV1(wt), PV1(F69H), and P3(wt). The mock controls are shown in lanes 2 and 3 of panels A and B, respectively;the guanidine HCl control reaction (described in the legend to Fig. 4) is shown in the lanes marked with asterisks. The amount of P3(wt) RNA added increased in rangefrom 0.9 to 7.2 nM of message (0.9 nM for lanes 4 and 8, 3.6 nM for lanes 5 and 9, and 7.2 nM for lanes 6 and 10 of panel A; 0.9 nM for lanes 5 and 9, 3.6 nM forlanes 6 and 10, and 7.2 nM for lanes 7 and 11 of panel B). The reactions shown in lanes 1 to 3 and 7 of panel A and lanes 2 to 4 and 8 of panel B did not receive anyRNA encoding P3(wt) mRNA.

7196 TOWNER ET AL. J. VIROL.

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 7: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

able to complement replication-defective lesions in regions of3AB previously thought to be cis acting (22).

Complementation by mutated versions of P3. In an effort tofurther define the mechanism of P3(wt) rescue, mutated formsof P3 containing lethal mutations in the putative nucleoside

triphosphate binding site of the PV RNA polymerase 3D(3D*) or in the proteolytic active site of the proteinase 3C(3C*) were tested. Both of these mutations (K61L in 3D andC147A in 3C) render these proteins completely inactive fortheir polymerase (elongation) (38) and proteinase (30) activi-

FIG. 6. trans-dominant inhibition of RNA replication by P3(F69H). The overall format is like that described in the legend to Fig. 2 except that the RNAs used toprogram each parallel translation and replication reaction are PV1(wt), P3(wt), and P3(F69H). The mock controls are shown in lanes 2 and 3 of panels A and B,respectively; the guanidine HCl control reaction (described in the legend to Fig. 4) is shown in the lanes denoted by asterisks. The amounts of P3 mRNA added to eachreaction increased in range from 1.8 to 7.2 nM of message (1.8 nM for lanes 4 and 7, 3.6 nM for lanes 5 and 8, and 7.2 nM for lanes 6 and 9 of panel A; 1.8 nM forlanes 5 and 8, 3.6 nM for lanes 6 and 9, and 7.2 nM for lanes 7 and 10 of panel B). The reactions shown in lanes 1 to 3 of panel A and lanes 2 to 4 of panel B didnot receive any P3(wt) or P3(F69H) mRNA.

FIG. 7. Differential complementation by mutated versions of P3. The overall format is like that described in the legend to Fig. 2 except that the RNAs used toprogram each parallel translation and replication reaction are PV1(wt), PV1(F69H), P3(wt), P3(3D*), and P3(3C*). P3(3D*) contains a K61L mutation that rendersthe polymerase inactive for chain elongation, and P3(3C*) contains a C147A mutation that renders the 3C proteinase domain inactive. The mock controls are shownin lanes 2 and 3 of panels A and B, respectively; the guanidine HCl control reaction (described in the legend to Fig. 4) is shown in the lanes denoted by asterisks. Theamounts of P3 RNAs added were all at the same concentration of 3.6 nM (lanes 4 to 6 and 8 to 13 of panel A; lanes 5 to 7 and 9 to 11 of panel B). The reactions shownin lanes 1 to 3 and 7 of panel A and lanes 2 to 4 and 8 of panel B did not receive any P3-encoding RNA. Note that P3(3C*) (panel A, lane 13) shows no proteolyticcleavage, unlike P3(wt) and P3(3D*) (panel A, lanes 11 and 12).

VOL. 72, 1998 trans COMPLEMENTATION BY A POLIOVIRUS POLYPROTEIN 7197

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 8: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

ties. The requirement for a physical linkage between the activeviral polymerase and the putative carrier of VPg during RNAreplication complex formation was tested by programmingin vitro translation/replication reactions with PV1(wt) andPV1(F69H) mRNAs in the presence and absence of P3(3D*)or P3(wt) (Fig. 7A, lanes 3 to 5 and 7 to 9). A clear increase insome P3-derived proteins (e.g., 3CD) can be seen in lanes 4, 5,8, and 9, but otherwise all levels of protein production appearequivalent. Translation of all three forms of P3 in the absenceof any PV1 RNA can be seen in Fig. 7A, lanes 11 to 13. Thecorresponding RNA replication reactions are shown in Fig. 7B,lanes 4 to 6 and 8 to 10. In this experiment, a slight decrease inRNA replication can be seen when P3(3D*) is present in thewild-type RNA replication reaction (compare lanes 5 and 6).When P3(3D*) is cotranslated with PV1(F69H), we observe asignificant rescue of RNA replication (Fig. 7B; compare lane 8to lane 10) that is slightly less than the level of rescue effectedby P3(wt) (Fig. 7B; compare lane 8 to lane 9). Overall, theseresults suggest that 3AB and the functional RNA polymerasedo not need to enter the complex as part of the same poly-protein molecule given that the active polymerase was de-rived from the PV1(F69H) RNA. Furthermore, the abilityof P3(3D*) to partially poison the PV1(wt) replication com-plex and diminish the rescue of the PV1(F69H) replicationdefect is consistent with previous studies suggesting that 3Dpol

can be provided in trans to the viral RNA replication machin-ery (14, 34).

To determine if intact P3 provides a replication functiondistinct from that of cleaved P3, the ability of a noncleaving P3molecule [P3(3C*)] to rescue PV1(F69H) was assayed. Theresults of the translation assays are shown in Fig. 7A, lanes 6and 10; those for the RNA synthesis reactions are shown inFig. 7B, lanes 7 and 11. Comparison of lane 8 to lane 11 of Fig.7B indicates that PV1(F69H) RNA replication was not res-cued, suggesting that the F69H lesion does not act at the levelof the entire P3 polyprotein. An interesting observation is thatsimilar amounts of 3AB(wt) are generated from all three P3molecules tested (Fig. 7A, lanes 8 to 10). Considering thatP3(3C*) cannot cleave either in cis or in trans to generate 3AB,the generation of 3AB from the cotranslated P3 moleculesmust result from trans cleavage by proteinases derived from thefull-length PV1 RNA.

DISCUSSION

The data presented in this study show that wild-type P3polypeptide can trans complement a PV RNA replication de-fect caused by a single amino acid substitution (F69H) in thehydrophobic domain of protein 3AB. Surprisingly, no comple-mentation was seen when we performed similar experiments inwhich 3AB(wt) was similarly provided in trans. The trans-res-cue phenotype of P3(wt) is further corroborated by experi-ments in which P3 proteins containing the same F69H lesionexert substantial trans-dominant effects on a wild-type RNAreplication complex. No such inhibition is observed when thesame F69H mutation is present in the smaller P3 cleavageproduct 3AB and tested under similar conditions. In addition,the ability of P3(3D*) to successfully trans complement thedefective replication complex indicates that the active RNApolymerase 3Dpol and 3AB (or 3A or 3ABC) need not bedelivered to the replication complex from the same molecule.This result suggests that the mechanism of complementation isnot as simple as providing more RNA polymerase to a func-tional but crippled replication complex since the extra RNApolymerase is itself nonfunctional.

Based on these results, the RNA replication defect in pro-

tein 3AB may be complemented because P3 provides a pre-cursor molecule(s) to VPg. The precursor to VPg has beenhypothesized to be 3AB (40, 43, 44), a lipophilic protein capa-ble of tight membrane association (47, 52). Utilization of thismechanism would indicate that P3 supplies 3AB, or possibly analternative VPg-containing precursor, and that the role of P3in primary complex formation is to merely deliver the neces-sary VPg-containing P3 cleavage product. This latter hypoth-esis is consistent with all of the data presented in our study,including the observation that P3(3D*) and P3(wt) are able torescue PV1(F69H).

It is possible that protein 3AB is not the true precursor ofVPg. Rather, the precursor may be another VPg-containingpolyprotein such as 3BCD or 3ABC (Fig. 1). If the VPg pre-cursor is 3BCD, the data suggest that (i) 3BCD is not theprecursor to the 3D RNA polymerase [due to the ability ofP3(3D*) to complement] and (ii) the proposed anchoring func-tion of 3A would be mediated through tight protein contactsand not via covalent attachment to 3B or other 3B-containingproteins. An observation that favors 3BCD (or 3BC) as theimmediate precursor of VPg is that cleavage of in vitro-trans-lated P3(wt) at the 3A-3B junction is dilution independentwhereas cleavage of the 3B-3C junction is dilution dependent(53). These data are consistent with the inability of P3(3C*) tocomplement RNA synthesis of the 3A(F69H) lesion becausethis proteolytically inactive form of P3 would be unable tocleave in cis to generate 3BCD. However, it would be a suitablesubstrate for a dilution-dependent (trans) cleavage to generatethe 3AB observed in the in vitro translation (Fig. 7A, lane 10).

Complementation of a lethal VPg mutation by 3AB(wt) wasobserved by Cao and Wimmer (13), who provided 3AB in transby placing the 3AB coding sequence in the first cistron of adicistronic RNA. The virus generated was genetically unstable,undergoing multiple genetic rearrangements. The results ofthis unique approach are difficult to compare directly to theresults presented here; however, the fact that any virus wasrecovered at all is indicative of complementation. It shouldalso be pointed out that the exact details of the defect con-ferred by the 3AB(F69H) mutation are not known, and there-fore it is possible that the mechanism of VPg donation may notbe perturbed at all, but that an alternative function of 3AB (ora larger 3A-containing polyprotein) is affected. The possibilitythat 3AB is involved in multiple functions is supported by theability to complement an amino acid insertion in the N-termi-nal half of 3A (6) by using a helper virus, while a temperature-sensitive mutation in the hydrophobic domain of 3AB couldnot be similarly complemented (22).

There is precedent for precursor polypeptide functions dis-tinct from those of mature cleavage products in the RNAreplication activities of other positive-strand RNA viruses. Al-phaviruses like Sindbis virus and Semliki Forest virus encodetheir replication proteins (including nsP4, the putative RNApolymerase) in the form of polyproteins encoded in the 59portion of their genomic RNAs. Data from cell culture studiesusing mutant viruses (or plasmids that encode RNAs withsite-directed lesions) showed that the mature nsP4 polymeraseand an uncleaved precursor polypeptide (P123) are requiredfor synthesis of negative-strand intermediates in Sindbis virus-infected cells. However, virus-specific proteolytic processing ofthis P123 precursor polypeptide effects a switch from negative-strand RNA synthesis to positive-strand synthesis (31, 41).Based on these observations, one might speculate that the PVP3 replicase precursor is required for initiation of the syn-thesis of negative-strand intermediates and that mature 3ABpolypeptides or other cleavage products function in the syn-

7198 TOWNER ET AL. J. VIROL.

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 9: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

thesis of positive-strand RNAs by using these newly synthe-sized intermediates as templates.

A model for replication complex assembly is depicted in Fig.8. Figure 8A shows a scenario in which the PV proteins arefully processed prior to complex assembly. This model predictsthat all of the freely interchangeable viral proteins capable ofcomplementation are mature cleavage products. An alterna-tive mechanism for complex assembly is shown in Fig. 8B. Inthis model, the RNA replication function disrupted by the3AB(F69H) mutation is initially delivered to the replicationcomplex by the P3 precursor protein. As a result, the mecha-nism of replication complex assembly would involve a multi-step process in which the mature virus proteins are properlypositioned only when delivered from defined precursor pro-teins in a prerequisite assembly step. This idea, together withthe intricate protein processing cascade that generates themature PV proteins, underscores the notion that the replica-tion complex is an active and kinetically transient structure.The model in Fig. 8B does not rule out the ability of otherfunctions of the same protein to interchange as mature viralcleavage products, an important consideration given that someof the nonstructural proteins have been shown to be both cisand trans acting.

In summary, this study provides important clues as to howthe PV RNA replication complex may initially assemble. Muchof the previous complementation data that suggested require-ment for a viral protein acting in cis could be explained by theassembly mechanism outlined in Fig. 8B. The replication com-plex consists of a combination of mature and stable virus cleav-age products mixed with viral protein precursors with shorthalf-lives (in addition to template RNAs, possibly cellular pro-teins, and membranes). If a cis-acting lesion in a virus proteinexerts its effects at the level of a transient precursor protein,then this model of replication complex assembly predicts thatthe kinetic window for genetic complementation will be limiteddue to the requirement of a series of ordered subunit interac-

tions. In addition, the concentrations of such precursors willnever reach (or sustain) the levels of the mature viral proteins,thereby limiting the complementation potential of a given pre-cursor polypeptide. Based on this hypothesis, complementa-tion might be enhanced by using PV mutants with retardedproteolytic processing kinetics where precursor assembly inter-mediates would have longer half-lives. Our model also predictsthat when successful complementation does occur, the defec-tive replication function must be rescued by an interchange-able protein unit. In the case of protein 3AB, the data suggestthat the interchangeable protein unit is P3 and not the maturepolypeptide.

ACKNOWLEDGMENTS

We are grateful to Todd Parsley and Stacey Stewart for criticalcomments on the manuscript and to Ollie Richards for the generousgift of the plasmid containing the m61 lesion in 3Dpol.

This work was supported by Public Health Service grant AI 22693from the National Institutes of Health and by services provided by theIMAGE facility of the School of Biological Sciences, University ofCalifornia, Irvine.

REFERENCES1. Alexander, L., H. H. Lu, and E. Wimmer. 1994. Polioviruses containing

picornavirus type 1 and/or type 2 internal ribosome entry site elements:genetic hybrids and the expression of a foreign gene. Proc. Natl. Acad. Sci.USA 91:1406–1410.

2. Andino, R., G. E. Rieckhof, P. L. Achacoso, and D. Baltimore. 1993. Polio-virus RNA synthesis utilizes an RNP complex formed around the 59-end ofviral RNA. EMBO J. 12:3587–3598.

3. Andino, R., G. E. Rieckhof, and D. Baltimore. 1990. A functional ribonucle-oprotein complex forms around the 59 end of poliovirus RNA. Cell 63:369–380.

4. Banerjee, R., A. Echeverri, and A. Dasgupta. 1997. Poliovirus-encoded 2Cpolypeptide specifically binds to the 39-terminal sequences of viral negative-strand RNA. J. Virol. 71:9570–9578.

5. Barton, D. J., E. P. Black, and J. B. Flanegan. 1995. Complete replication ofpoliovirus in vitro: preinitiation RNA replication complexes require solublecellular factors for the synthesis of VPg-linked RNA. J. Virol. 69:5516–5527.

6. Bernstein, H. D., P. Sarnow, and D. Baltimore. 1986. Genetic complemen-

FIG. 8. Schematic representation of proposed mechanisms of replication complex assembly. (A) Following translation, viral proteins are co- and posttranslationallyprocessed to generate mature virus proteins. The mature virus proteins then form the functional replication complex that is associated with cytoplasmic membranestructures (depicted as ladder-like forms). (B) Following translation, viral proteins undergo limited proteolytic cleavage, generating the larger precursor protein P3and/or other precursor proteins such as 2BC-P3. These larger precursor proteins then interact to form a complex which subsequently undergoes further proteolyticcleavages to generate a functional RNA replication complex. As shown in this model, P3 is the precursor protein from which 3AB is derived.

VOL. 72, 1998 trans COMPLEMENTATION BY A POLIOVIRUS POLYPROTEIN 7199

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

Page 10: Rescue of Defective Poliovirus RNA Replication by 3AB-Containing

tation among poliovirus mutants derived from an infectious cDNA clone.J. Virol. 60:1040–1049.

7. Bernstein, H. D., and D. Baltimore. 1988. Poliovirus mutant that contains acold-sensitive defect in viral RNA synthesis. J. Virol. 62:2922–2928.

8. Bienz, K., D. Egger, T. Pfister, and M. Troxler. 1992. Structural and func-tional characterization of the poliovirus replication complex. J. Virol. 66:2740–2747.

9. Brown, B. A., and E. Ehrenfeld. 1979. Translation of poliovirus RNA in vitro:changes in cleavage pattern and initiation sites by ribosomal salt wash.Virology 97:396–405.

10. Caliguiri, L. A., and I. Tamm. 1968. Action of guanidine on the replicationof poliovirus RNA. Virology 35:408–417.

11. Caliguiri, L. A., and I. Tamm. 1970. Characterization of poliovirus-specificstructures associated with cytoplasmic membranes. Virology 42:112–122.

12. Caliguiri, L. A., and I. Tamm. 1970. The role of cytoplasmic membranes inpoliovirus biosynthesis. Virology 42:100–111.

13. Cao, X., and E. Wimmer. 1995. Intragenomic complementation of a 3ABmutant in dicistronic polioviruses. Virology 209:315–326.

14. Charini, W. A., C. C. Burns, E. Ehrenfeld, and B. L. Semler. 1991. transrescue of a mutant poliovirus RNA polymerase function. J. Virol. 65:2655–2665.

15. Cho, M. W., N. Teterina, D. Egger, K. Bienz, and E. Ehrenfeld. 1994.Membrane rearrangement and vesicle induction by recombinant poliovirus2C and 2BC in human cells. Virology 202:129–145.

16. Collis, P. S., B. J. O’Donnell, D. J. Barton, J. A. Rogers, and J. B. Flanegan.1992. Replication of poliovirus RNA and subgenomic RNA transcripts intransfected cells. J. Virol. 66:6480–6488.

17. Datta, U., and A. Dasgupta. 1994. Expression and subcellular localization ofpoliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for gly-cosylation in vitro. J. Virol. 68:4468–4477.

18. Dewalt, P. G., and B. L. Semler. 1989. Molecular biology and genetics ofpoliovirus protein processing, p. 73–93. In B. L. Semler and E. Ehrenfeld(ed.), Molecular aspects of picornavirus infection and detection. AmericanSociety for Microbiology, Washington, D.C.

19. Dugal, R., A. Cuconati, M. Gromeier, and E. Wimmer. 1997. Genetic re-combination of poliovirus in a cell-free system. Proc. Natl. Acad. Sci. USA94:13786–13791.

20. Echeverri, A. C., and A. Dasgupta. 1995. Amino terminal regions of polio-virus 2C protein mediate membrane binding. Virology 208:540–553.

21. Gamarnik, A. V., and R. Andino. 1997. Two functional complexes formed byKH domain containing proteins with the 59 noncoding region of poliovirusRNA. RNA 3:882–892.

22. Giachetti, C., S.-S. Hwang, and B. L. Semler. 1992. cis-acting lesions targetedto the hydrophobic domain of a poliovirus membrane protein involved inRNA replication. J. Virol. 66:6045–6057.

23. Haller, A. A., and B. L. Semler. 1992. Linker scanning mutagenesis of theinternal ribosome entry site of poliovirus RNA. J. Virol. 66:5075–5086.

24. Harris, K. S., W. Xiang, L. Alexander, W. S. Lane, A. V. Paul, and E.Wimmer. 1994. Interaction of poliovirus polypeptide 3CDpro with the 59 and39 termini of the poliovirus genome. Identification of viral and cellularcofactors needed for efficient binding. J. Biol. Chem. 269:27004–27014.

25. Hope, D. A., S. E. Diamond, and K. Kirkegaard. 1997. Genetic dissection ofinteraction between poliovirus 3D polymerase and viral protein 3AB. J.Virol. 71:9490–9498.

26. Johnson, K. L., and P. Sarnow. 1991. Three poliovirus 2B mutants exhibitnoncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus. J. Virol. 65:4341–4349.

27. Kuge, S., and A. Nomoto. 1987. Construction of viable deletion and insertionmutants of the Sabin strain of type 1 poliovirus: function of the 59 noncodingsequence in viral replication. J. Virol. 61:1478–1487.

28. Kuge, S., I. Saito, and A. Nomoto. 1986. Primary structure of poliovirusdefective-interfering particle genomes and possible generation mechanismsof the particles. J. Mol. Biol. 192:473–487.

29. Lama, J., A. V. Paul, K. S. Harris, and E. Wimmer. 1994. Properties ofpurified recombinant poliovirus protein 3AB as substrate for viral proteinasesand as co-factor for RNA polymerase 3Dpol. J. Biol. Chem. 269:66–70.

30. Lawson, M. A., and B. L. Semler. 1991. Poliovirus thiol proteinase 3C can

utilize a serine nucleophile within the putative catalytic triad. Proc. Natl.Acad. Sci. USA 88:9919–9923.

31. Lemm, J. A. T. Rumenapf, E. G. Strauss, J. H. Strauss, and C. M. Rice. 1994.Polypeptide requirements for assembly of functional Sindbis virus replica-tion complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis. EMBO J. 13:2925–2934.

32. Li, J. P., and D. Baltimore. 1988. Isolation of poliovirus 2C mutants defectivein viral RNA synthesis. J. Virol. 62:4016–4021.

33. Lundquist, R. E., and J. V. Maizel. 1978. In vivo regulation of the poliovirusRNA polymerase. Virology 89:484–493.

34. Novak, J. E., and K. Kirkegaard. 1994. Coupling between genome transla-tion and replication in an RNA virus. Genes Dev. 8:1726–1737.

35. Parsley, T. B., and B. L. Semler. 1998. Unpublished observations.36. Parsley, T. B., J. S. Towner, L. B. Blyn, E. Ehrenfeld, and B. L. Semler. 1997.

Poly (rC) binding protein 2 forms a ternary complex with the 59-terminalsequences of poliovirus RNA and the viral 3CD proteinase. RNA 3:1124–1134.

37. Plotch, S. J., and O. Palant. 1995. Poliovirus protein 3AB forms a complexwith and stimulates the activity of the viral RNA polymerase, 3Dpol. J. Virol.69:7169–7179.

38. Richards, O. C., S. Baker, and E. Ehrenfeld. 1996. Mutation of lysine resi-dues in the nucleotide binding segments of the poliovirus RNA-dependentRNA polymerase. J. Virol. 70:8564–8570.

39. Roehl, H. H., and B. L. Semler. 1995. Poliovirus infection enhances theformation of two ribonucleoprotein complexes at the 39 end of viral negative-strand RNA. J. Virol. 69:2954–2961.

40. Semler, B. L., C. W. Anderson, R. Hanecak, L. F. Dorner, and E. Wimmer.1982. A membrane-associated precursor to poliovirus VPg identified byimmunoprecipitation with antibodies directed against a synthetic heptapep-tide. Cell 28:405–412.

41. Shirako, Y., and J. H. Strauss. 1994. Regulation of Sindbis virus RNAreplication: uncleaved P123 and nsP4 function in minus-strand RNA synthe-sis, whereas cleaved products from P123 are required for efficient plus-strandRNA synthesis. J. Virol. 68:1874–1885.

42. Summers, D. F., and J. V. Maizel. 1968. Evidence for large precursor pro-teins in poliovirus synthesis. Proc. Natl. Acad. Sci. USA 59:966–971.

43. Takeda, N., R. J. Kuhn, C. F. Yang, T. Takegami, and E. Wimmer. 1986.Initiation of poliovirus plus-strand RNA synthesis in a membrane complex ofinfected HeLa cells. J. Virol. 60:43–53.

44. Takegami, T., R. J. Kuhn, C. W. Anderson, and E. Wimmer. 1983. Mem-brane-dependent uridylylation of the genome-linked protein VPg of polio-virus. Proc. Natl. Acad. Sci. USA 80:7447–7451.

45. Takegami, T., B. L. Semler, C. W. Anderson, and E. Wimmer. 1983. Mem-brane fractions active in poliovirus RNA replication contain VPg precursorpolypeptides. Virology 128:33–47.

46. Tang, R. S., D. J. Barton, J. B. Flanegan, and K. Kirkegaard. 1997. Polio-virus RNA recombination in cell-free extracts. RNA 3:624–633.

47. Tershak, D. R. 1984. Association of poliovirus proteins with the endoplasmicreticulum. J. Virol. 52:777–783.

48. Teterina, N. L., A. E. Gorbalenya, D. Egger, K. Bienz, and E. Ehrenfeld.1997. Poliovirus 2C protein determinants of membrane binding and rear-rangements in mammalian cells. J. Virol. 71:8962–8972.

49. Teterina, N. L., W. D. Zhou, M. W. Cho, and E. Ehrenfeld. 1995. Inefficientcomplementation activity of poliovirus 2C and 3D proteins for rescue oflethal mutations. J. Virol. 69:4245–4254.

50. Todd, S., J. H. C. Nguyen, and B. L. Semler. 1995. RNA-protein interactionsdirected by the 39 end of human rhinovirus genomic RNA. J. Virol. 69:3605–3614.

51. Todd, S., J. S. Towner, and B. L. Semler. 1997. Translation and replicationproperties of the human rhinovirus genome in vivo and in vitro. Virology229:90–97.

52. Towner, J. S., T. V. Ho, and B. L. Semler. 1996. Determinants of membraneassociation for poliovirus protein 3AB. J. Biol. Chem. 271:26810–26818.

53. Towner, J. S., and B. L. Semler. 1998. Unpublished observations.54. Xiang, W., K. S. Harris, L. Alexander, and E. Wimmer. 1995. Interaction

between the 59-terminal cloverleaf and 3AB/3CDpro of poliovirus is essentialfor RNA replication. J. Virol. 69:3658–3667.

55. Xiang, W., and E. Wimmer. 1997. Unpublished observations.

7200 TOWNER ET AL. J. VIROL.

on Novem

ber 22, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from


Recommended