Evidence for Functional Protein Interactions Required for Poliovirus RNA Replication

doi:10.1128/JVI.02684-05

J Virol. 2006 June; 80(11): 5327–5337.

PMCID: PMC1472133

Evidence for Functional Protein Interactions Required for Poliovirus RNA Replication

Natalya L. Teterina,¹ Eric Levenson,¹ Mario S. Rinaudo,¹ Denise Egger,² Kurt Bienz,² Alexander E. Gorbalenya,³ and Ellie Ehrenfeld¹^*

Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland 20892,¹ Institute for Medical Microbiology, University of Basel, Basel, Switzerland,² Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands³

^*Corresponding author. Mailing address: LID, NIAID, NIH, Bldg. 50, Room 6120, 50 South Drive, Bethesda, MD 20892-8011. Phone: (301) 594-1654. Fax: (301) 435-6021. E-mail: eehrenfeld/at/niaid.nih.gov.

Received December 21, 2005; Accepted March 8, 2006.

This article has been cited by other articles in PMC.

Abstract

Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell two-hybrid analysis.

Poliovirus (PV) RNA replication takes place in replication complexes that form de novo in cultured cells after virus infection. Synthesis of viral proteins induces extensive rearrangement of intracellular membrane structures that produce perinuclear foci of vesicle-associated viral proteins and RNA (reference 11 and references therein). These coalesce into large clusters of vesicles engaged in viral RNA synthesis, accompanied by the loss of preexisting Golgi stacks and endoplasmic reticulum (ER). All viral nonstructural proteins, derived from the P2 and P3 polyprotein regions of the single open reading frame in the viral genome, are found associated with the membranous replication complexes and have been implicated by genetic analysis as playing essential roles in the process of viral RNA replication. Proteins containing 2B, 2C, or 3A sequences manifest inherent membrane-binding properties. It is not known how the other viral proteins are recruited to and/or retained in the replication complexes. They may enter or induce formation of the complexes as larger precursor proteins prior to cleavage and maintain their associations via protein-protein or protein-RNA interactions, a hypothesis supported by the observation that complementation of defective proteins by expression of individual functional gene products does not occur readily in infected cells (32) and requires expression of whole P2 or P3 precursor proteins in vitro (16, 35).

The precise biochemical roles in viral RNA synthesis played by each of the nonstructural proteins are poorly defined (summarized in reference 21). From the P3 region, protein 3D catalyzes polynucleotide chain elongation as well as uridylylation of VPg (protein 3B) to form a primer for RNA chain initiation. Protein 3C is the protease responsible for the majority of polyprotein cleavages, both in cis and in trans. Protein 3CD, in addition to serving a proteinase function for generation of capsid proteins from P1 precursors, binds and stimulates utilization of cis-acting viral RNA structures, both at the 5′-terminal cloverleaf structures required for RNA chain initiation and at the internal stem-loop structure that presents the template for uridylylation of VPg. Protein 3A is a trans-membrane-binding protein that inhibits ER-to-Golgi membrane and secretory protein traffic (7, 8).

From the P2 region, 2A may have an unidentified function in viral RNA synthesis, 2B contains a membrane-binding region, and viral protein 2C and precursors containing 2C sequences are tightly associated with the remodeled membranous structures. 2BC, alone or in conjunction with protein 3A, is considered to be responsible for inducing intracellular membrane reorganization (4, 26, 28). As such, 2C or 2B sequences are common localization markers for the viral RNA replication complex.

Although the atomic structure of protein 2C has not been resolved, comparative sequence and bioinformatics analyses have predicted a three-domain structure, of which the central domain is most highly conserved among picornaviruses and other small RNA and DNA viruses (30). This central portion contains nucleoside triphosphate-binding and predicted helicase motifs (13), and ATPase activity has been demonstrated for purified recombinant 2C fusion proteins (19, 23, 25). Mutations in this domain that confer sensitivity or resistance of the virus to mM concentrations of guanidine-HCl have been shown to affect 2C's ATPase activity (23). Both the N- and C-terminal regions manifest RNA-binding activity (1, 24). They can be folded into amphipathic α-helical structures (22), and the N-terminal region appears to be the primary determinant of the protein's membrane-binding property (9, 10, 30). The capacity to form an N-terminal amphipathic helix is conserved among all picornaviruses examined, and some mutations predicted to disrupt the helical fold are detrimental to viral RNA synthesis (22). The 2C protein has been implicated in additional viral functions, such as virion assembly (17, 36), although no specific domain assignments have been made for these other functions.

Previous studies from this laboratory were aimed at analyzing the viability and growth patterns of chimeric polioviruses in which the N-terminal amphipathic helix in viral protein 2C was replaced by orthologous sequences from other picornaviruses or from the NS5A protein from hepatitis C virus (31). Chimeric virus containing sequences encoding the 2C amphipathic helix from human rhinovirus (HRV) type 14 (HRV14) harbors seven amino acid changes from the core of 18 residues in the PV 2C amphipathic helix. The virus was viable but exhibited defects in viral RNA replication that led to delayed and reduced yields of viral RNA and produced minute plaques on HeLa cell monolayers. Among the small plaques, large plaque variants appeared with a relatively high frequency. Some of these contained virus in which either of two single HRV14-specific amino acid residues, A22 or L28, had reverted to the S22 or F28 (or S28) normally present in PV strains, suggesting that these two residues might be involved in specific protein contacts. Viruses with improved growth properties also were isolated with other single amino acid changes in 2C outside of the region of the amphipathic helix. However, the majority of viruses that emerged with improved growth properties (approximately 60%) contained no reversions within the HRV amphipathic helix-encoding sequence or anywhere else in the region encoding 2C. In the present study, we performed sequence analyses on these pseudorevertant viral genomes and reconstructed viruses with individual mutations found in either 3A or 2B sequences which compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins. These genetic data suggested that the N-terminal amphipathic helix in protein 2C participates either directly or indirectly in molecular interactions with sequences from at least two different viral proteins and provided evidence for the functional relevance of these interactions. Binding among these viral proteins was confirmed by mammalian two-hybrid analysis.

MATERIALS AND METHODS

Construction of plasmids and DNA manipulation.

Primer sequences used in PCR to clone wild-type and mutant PV sequences into various vectors are available on request. Standard recombinant DNA technology was used to construct and purify all plasmid DNAs. The integrity of all PCR-derived DNA fragments was verified by automated DNA sequencing with an ABI 3100 gene analyzer (Applied Biosystems). A BigDye Terminator version 3.1 sequencing kit was used according to the manufacturer's instructions. Plasmid DNAs were purified from bacterial cultures by use of a QIAGEN Plasmid Maxi kit (Valencia, CA). All nucleotide numbers refer to the PV type 1 (PV1) genome, and all amino acid numbers refer to the indicated PV protein (e.g., 2B-T35A).

The plasmid pT7PV1 (14) contains a full-length infectious cDNA of PV1. The modified full-length plasmid pT7PVxx, the plasmid pPV-2Cah-HRV14, encoding the N-terminal amphipathic helix in 2C from HRV14, and plasmid pPV-2Cah-HRV14-L28F have been previously described (31).

Plasmids pPV-μ2B, pPV-2Cah-HRV14-μ2, pPV-μ3A, and pPV-2Cah-HRV14-μ3A, harboring mutations encoding substitutions of 2B-T35A and 3A-K9R in PVxx or PV-2Cah-HRV14 genomes, respectively, were generated by site-directed mutagenesis performed with a QuikChange mutagenesis kit (Stratagene) and standard recombinant DNA technologies.

Plasmids pAct, pBind, and pG5luc were from the Checkmate mammalian two-hybrid system (Promega). The pAct plasmid contains the herpes simplex virus type 1 VP16 activation domain (Act), and the pBind plasmid contains the yeast GAL4 DNA-binding domain (Bind). Both plasmids contain identical multiple cloning sites immediately downstream of Act or Bind. In addition, pBind plasmid carries the Renilla luciferase gene driven by an independent promoter, which allows monitoring of transfection efficiency. The pGal5luc reporter plasmid contains five GAL4-binding sites upstream of a minimal TATA box that precedes the firefly luciferase gene.

The DNA fragments encoding the PV proteins 2B, 2C, 2BC, 3A, and 3AB (wild type or mutant) were amplified by PCR using pT7PV1, pPV-2Cah-HRV14, pPV-2Cah-HRV14-2BT35A, pPV-2Cah-HRV14-L28F, or pPVxx-2C-F28L as the template. Primers used for PCR amplification introduced a SalI site at the upstream end and a stop codon plus an MluI site at the downstream end of amplified fragments. The pAct and pBind plasmids were digested with the same restriction enzymes, and PCR fragments were cloned in these vectors. The nucleotide sequences of all inserts were verified by sequence analysis. The plasmids are named after the parental plasmids and the viral protein (e.g., pBind2C). In the case of mutant viral proteins, mutations are also indicated (e.g., pBind2Cah-HRV14-L28F).

Transfection and two-hybrid analysis.

COS cells were grown in 12-well tissue culture plates to 95% confluence. Cells were transfected with a total of 1.6 μg plasmid DNA by use of Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions with the following modifications. In preliminary experiments, we used a 1:1:1 mix of pBind:pAct:pG5luc plasmids (0.53 μg of each). At these concentrations, we observed strong effects of one plasmid on expression from the other, presumably due to competition among promoters for transcription factors. After analysis of the effect of plasmid concentration on the expression of proteins and on the induction of luciferase expression, we selected optimized conditions that consisted of significantly lower amounts of pBind and pAct plasmids (0.053 μg pBind, 0.053 μg pAct; 1:1), with a higher concentration of pG5luc (1 μg) and an additional 0.49 μg of pGEM3 (used as carrier DNA for optimal transfection). For each transfection, 4 μl Lipofectamine 2000 reagent was added to 100 μl serum-free medium (Opti-MEM; Gibco) and incubated at room temperature for 15 min, after which the mixture of plasmid DNAs was diluted in 100 μl of serum-free medium (Opti-MEM) and added for an additional 15 min at room temperature. After incubation, the Lipofectamine-DNA mixture was added to the cells. Transfections were performed in triplicate. After 24 to 28 h of incubation at 37°C, luciferase activities were determined with the luciferase reporter assay system (Promega) as described by the manufacturer's protocol. Briefly, the cells were lysed in 250 μl of lysis buffer (Promega) and incubated for 15 min. Ten microliters of cellular extract was mixed with 100 μl of firefly luciferase substrate (Promega), and the luciferase-mediated light emission was measured as relative light units in a 1450 Microbeta (Wallac) luminometer.

Electron microscopy.

Cells were detached from the substrate, fixed with 2.5% glutaraldehyde and 1% OsO₄, and embedded in EPON 812. Sections were photographed in a Philips CM100 electron microscope.

RESULTS

Isolation of pseudorevertants of PV-2Cah-HRV14.

Figure Figure1A1A shows a schematic diagram of the chimeric poliovirus genome in which the sequence coding for the N-terminal amphipathic helix in protein 2C was replaced by the corresponding sequence from HRV14. Among the minute plaques produced by transfection of HeLa cell monolayers with this RNA, large plaques were observed with relatively high frequency (~10⁻²; see Fig. Fig.2B2B and reference 31).

FIG. 1.

Schematic diagrams of the chimeric PV genome encoding the N-terminal amphipathic helix in protein 2C from HRV14 and pseudorevertants. (A) The position of the sequence encoding the amphipathic helix is indicated by a black box, and the encoded amino acid (more ...)

FIG. 2.

Single amino acid changes in 2B and 3A sequences restore wild-type plaque phenotype to PV-2Cah-HRV14 RNAs. (A) Schematic diagram of chimeric viral transcripts encoding compensatory amino acid changes in either the 2B or the 3A protein. μ2B refers (more ...)

Ten such plaques were isolated and used for two additional rounds of passaging and plaque purification. For each of the 10 primary virus isolates, two to four plaques of third-passage virus were obtained and used to produce 30 individual virus stocks for further analysis. Initial sequence analyses indicated that 12 of these virus isolates acquired mutations in the sequence encoding protein 2C; these were reported and described elsewhere (31). The locations of these mutations are indicated in Fig. Fig.1B1B (R1 through R5). However, 18 analyzed virus stocks did not contain any sequence changes throughout the entire 2C-encoding region. The inability to find any nucleotide changes in 2C of these PV-2Cah-HRV14 isolates, which at the same time showed significantly improved growth properties, suggested the acquisition of a mutation(s) outside of the 2C-encoding region. RNA isolated from HeLa cells infected with individual plaque-purified virus stocks was sequenced within the region coding for proteins 2B through 3C. In addition, the entire genome from one virus isolate (R9; Table Table1)1) was sequenced.

TABLE 1.

Large plaque viral isolates with mutations outside of the 2C-encoding region

Table Table11 shows a summary of the mutations located outside of the 2C-encoding region found in the analyzed viral isolates with improved growth properties. The observed acquired mutations cluster in two regions (Fig. (Fig.1B),1B), encoding PV protein 2B (R6, R7, R8) or protein 3A (R9, R10, R11). In all cases shown, the HRV14 sequences encoding the N-terminal amphipathic helix in the chimeric 2C protein were retained. Among the 2B mutations, the most frequently found was mutation A3935G (R6), which was observed in five virus isolates that were progeny of two independent primary virus plaques. This mutation results in a threonine-to-alanine change in amino acid 35 of poliovirus protein 2B (2B-T35A). Other mutations in the 2B-encoding sequence were found in three other virus isolates, which were progeny of two primary plaques (R7 and R8 in Table Table1).1). C3942A caused a threonine-to-isoleucine change in residue 37 of 2B, in very close proximity to the change in R6, whereas A4103T caused a change from isoleucine to leucine at amino acid 91, near the C terminus of 2B. For the group of viruses harboring mutations in 3A, mutation A5136G (R9) caused a substitution of lysine to arginine at amino acid 9 (3A-K9R); this mutation was present in three virus isolates, which were progeny of the same primary virus plaque. These isolates harbored an additional silent mutation (A5212G) slightly downstream of A5136G; however, subsequent separation of the two mutations in the 3A-encoding sequence showed that only the A5136G mutation contributed to improved growth of the PV-2Cah-HRV14 chimera (see below). Other mutations in the 3A-encoding sequence were identified in two isolates encoding 3A-K39E (R10) and in one isolate encoding 3A-S48R (R11).

Changes in either 2B or 3A protein mediate improved growth of the PV-2Cah-HRV14 chimera.

To ensure that the large-plaque phenotypes were due solely to the observed sequence changes, individual mutations representing the most frequently found in each group were engineered into the parental pPV-2Cah-HRV14 as well as into wild-type pPV1 (Fig. (Fig.2A),2A), and RNA transcripts from the reconstructed plasmids were used to transfect HeLa cells. Figure Figure22 shows the resulting plaque morphologies induced by each of the reconstructed pseudorevertant RNAs. In the context of the wild-type PV genome sequence, neither mutation caused any detectable effect on plaque morphology. Mutation 2B-T35A (nucleotide [nt] A3935G) appeared to compensate fully for the poor growth of PV-2Cah-HRV14, as PV-2Cah-HRV14-μ2B generated plaques of the same size and appearance as wild-type PV. Mutation K9R in protein 3A (nt A5136G) also significantly improved growth of the chimeric virus, although plaques produced by PV-2Cah-HRV-μ3A were slightly smaller than those produced by wild-type PV RNA. The silent mutation, A5212G, which was also found in the R9 virus isolates, had no additional phenotypic effect when engineered alone or in combination with that encoding K9R.

Analysis of viral RNA synthesis.

To examine whether the point mutations in protein 2B or 3A directly affected replication of chimeric PV genomes during the first cycle of virus replication, we measured positive-strand viral RNA synthesis in HeLa cells following transfection with RNA transcripts. At various times posttransfection, total cellular RNA was isolated and accumulation of positive-strand PV RNA was measured by a one-cycle RNase protection assay (Fig. (Fig.3).3). As described previously (31), chimeric PV-2Cah-HRV14 genome RNA accumulated significantly more slowly than did wild-type RNA during the first 12.5 h after transfection. Replication of chimeric genome PV-2Cah-HRV14-μ3A, however, was significantly improved, and the kinetics of replication of the chimeric genome PV-2Cah-HRV14-μ2B was indistinguishable from that of the wild type, consistent with the observed increase in plaque size. Interestingly, these point mutations in either 2B or 3A protein caused a slight delay in the replication of wild-type viral genomes. The data indicate that mutations in 2B or 3A protein directly affect the replication of chimeric PV genomes coding for protein 2C with an N-terminal amphipathic helix from HRV14.

FIG. 3.

Accumulation of chimeric PV RNAs in transfected cells. (A) HeLa cell monolayers were transfected with 1 μg of the indicated RNA transcripts. Cells were harvested at the indicated times after transfection. Total cytoplasmic RNA isolated from approximately (more ...)

Protein-protein interactions.

Rescue of a defective RNA replication phenotype caused by mutation in one viral protein with additional compensating mutations in another viral protein implies that there is some functional interaction between the two proteins during the replication process. Previous studies using yeast or mammalian two-hybrid systems evaluated specific protein binding among picornavirus proteins within either the P2 (5, 6) or the P3 (42) region. To corroborate the interactions revealed by our genetic data, we employed a mammalian two-hybrid system to evaluate the binding between PV proteins containing 2B, 2C, and 3A sequences. To this end, PV proteins 2B, 2C, 2BC, 3A, and 3AB were expressed as fusion proteins with the DNA-binding domain (Bind) of GAL4 (Bind-2B, Bind-2C, Bind-2BC, Bind-3A, and Bind-3AB) or as fusion proteins with the transactivation domain (Act) of herpes simplex virus type 1 VP16 protein (Act-2B and Act-2C, etc). Expression of the individual fusion proteins was monitored by immunoblot analysis with anti-GAL serum for the Bind fusions (Fig. (Fig.4)4) or anti-2C, anti-2B, or anti-3A serum for the Act fusions (data not shown) of total proteins extracted from COS-1 cells transfected with individual expression plasmids. Each fusion protein was observed migrating in the gel as expected for its predicted molecular mass. We observed that expression levels of the different fusion proteins were variable and that Act-3AB expression in particular was always relatively poor.

FIG. 4.

Expression of the GAL4 DNA-binding domain fusion proteins. COS-1 cells were transfected with pBind constructs encoding the indicated GAL4 fusion proteins. At 26 h posttransfection, proteins from total cell lysates were prepared, separated by sodium dodecyl (more ...)

To assay for protein interactions, 25 pairs of fusion proteins including every combination of the two groups were expressed in COS-1 cells in the presence of reporter plasmid pG5luc, which contains a firefly luciferase gene driven by a GAL4 promoter that is activated after interaction between the two fusion proteins. Figure Figure55 shows luciferase production observed 26 h after transfection with either pBind-2C (Fig. 5A and B) or pBind-2BC (Fig. 5C and D) in combination with each pAct fusion construct.

FIG. 5.

Mammalian cell two-hybrid analysis of interactions of PV fusion proteins. COS cells were cotransfected with pBind-2C (A and B) or pBind-2BC (C and D), and different pAct plasmids that drive expression of 2B, 2C, 2BC, 3A, and 3AB fusion proteins and the (more ...)

The Bind-2C fusion protein showed positive interactions with Act-2BC and with Act-3A, generating luciferase signals 6- to 25-fold above the background levels obtained with an empty fusion partner (Fig. (Fig.5A).5A). Signals produced by coexpression of Bind-2C and Act-2C fusion proteins were reproducibly above background, albeit by only ~2-fold (Fig. (Fig.5B).5B). A similarly low but reproducible increase in luciferase expression was observed when Bind-2C was coexpressed with Act-2B fusion protein, although pAct-2B always generated a rather high background signal when coexpressed with an empty pBind vector. All of these interactions except 2C-3A also were observed in the opposite orientation, when 2C was fused to Act and the potential binding partners were fused with Bind (e.g., Fig. Fig.5D5D for Bind-2BC). No interaction of 2C with 3AB was observed (Fig. (Fig.5B),5B), possibly because Act-3AB expression was quite low. Interaction between these two proteins also was not detected between fusions made in the opposite orientation, however.

The Bind-2BC fusion protein produced the largest increase in firefly luciferase activity when coexpressed with Act-2BC (~20-fold above background), indicating efficient homo-oligomerization of the 2BC protein (Fig. (Fig.5C)5C) similar to that demonstrated in previous studies (5, 6). Positive luciferase gene activation also resulted from coexpression of Bind-2BC with Act-3AB (~6.5-fold), despite low expression of the latter, with Act-3A (~7-fold), with Act-2C (~2.5-fold), and with Act-2B (~2.7-fold) (Fig. (Fig.5D).5D). The data for all tested protein pairs are summarized qualitatively in Table Table2.2. We arbitrarily defined luciferase signals greater than fourfold above background as indicative of positive interactions and considered two- to threefold increases in luciferase signals as possibly indicative of interactions (Table (Table2).2). It is not possible to compare the relative strengths or affinities of the different protein interactions on the basis of the amounts of luciferase activity induced. Although we devoted much effort to adjusting the assay conditions to avoid competition among promoters for transcription factors and to reduce the dependence of luciferase expression on plasmid concentration, the expression levels for different fusion proteins are not always the same, and the different fusion proteins may differentially affect the activities of the Act or Bind domains. Thus, the relative amounts of luciferase activity induced by unrelated pairs of hybrid proteins do not directly reflect differences in the strengths of interaction of the pairs of proteins. Qualitative inspection of the data in Table Table22 shows that in our assays all proteins tested except 3AB formed homomultimers. In addition, protein interactions were observed among most of the protein pairs tested, with the exception of 3AB-2C. As is frequently observed in two-hybrid analyses of protein interactions, we observed differences in luciferase activity induced by several pairs of proteins, depending on the polarity in which the two proteins were presented (e.g., compare 2C-2BC pair in Fig. 5A and D and and2B2B-2BC in Table Table22).

TABLE 2.

Linkage map of protein-protein interactions

The extent and complexity of interactions among proteins from the P2 and P3 regions of the poliovirus genome were not unexpected, since all the proteins examined are known to be colocalized and function within the viral RNA replication complex in infected cells. It should be noted that the failure to observe an interaction in the two-hybrid system does not necessarily mean that two proteins do not interact. Improper folding of the hybrid proteins can abrogate potential interactions and thus fail to activate transcription of the reporter gene in this assay. On the other hand, observation of induced luciferase activity in the presence of a pair of hybrid proteins is a strong indication that two proteins expressed as fusion polypeptides do interact.

Effect of 2C amphipathic helix on protein interactions.

It was of interest to determine whether the replacement of the N-terminal amphipathic helix in protein 2C by the orthologous element from HRV14 affected any of the protein-protein interactions described above and what effect the compensatory changes in protein 2B, 2C, or 3A had on these interactions. To address this question, we generated several additional constructs to express chimeric proteins 2Cah-HRV14 and 2BCah-HRV14 or these chimeric proteins bearing one of the mutations found in pseudorevertants of PV-2Cah-HRV14 fused to either the GAL4-binding domain or VP16 activation domain. We first examined the effects of the HRV14 amphipathic helix substitution in PV 2C and 2BC proteins on the formation of homo- and heterodimers observed with 2C, 2BC, 2B, and 3A proteins. Interactions of these mutant forms of 2C and 2BC fusion proteins with the 3AB protein were not analyzed because of the weak luciferase signals induced by 3AB reactions. Comparison of homodimerization and heterodimerization reactions of 2Cah-HRV14 and 2BCah-HRV14 fusion proteins with those observed for wild-type Bind-2C, Act-2C, Bind-2BC, and Act-2BC demonstrated that most pairs induced very similar levels of firefly luciferase activity (data not shown). This result was not unexpected, since regions of the proteins other than the amphipathic helix may be responsible for the interactions. There were, however, two exceptions. As shown in Fig. Fig.6A,6A, coexpression of Bind-2Cah-HRV14 and Act-2Cah-HRV14 induced luciferase activity approximately twofold higher than that induced by coexpression of the wild-type fusion proteins. This increase was reproduced in multiple independent experiments. The expression levels of mutant and wild-type proteins were carefully monitored within parallel experiments to exclude the possibility that differences in the induction of luciferase activity were merely due to variable expression of these proteins (data not shown). Thus, it appears that 2Cah-HRV14 protein exhibits a greater propensity to oligomerize than does wild-type 2C (Fig. (Fig.6A).6A). The single amino acid change in the amphipathic helix from HRV14 (L28F), which was previously shown to completely restore the replication phenotype of PV-2Cah-HRV14 (31), was sufficient to decrease the signal produced by the 2C homodimerization to a level comparable to that of wild-type 2C proteins (Fig. (Fig.6A6A).

FIG. 6.

Effect of the HRV14 amphipathic helix and compensatory mutations in PV protein 2C on protein interactions. (A) Mammalian two-hybrid analysis of the effect of substitutions in N-terminal amphipathic helix in protein 2C on homomultimerization. The cotransfected (more ...)

A lesser but reproducible increase in the interaction of Bind-2Cah-HRV14 fusion protein with Act-2BCah-HRV14 compared with wild-type fusion proteins was also observed; however, the differences were small and therefore were not pursued experimentally.

A second interaction observed to be affected by substitution of the N-terminal amphipathic helix in protein 2C was that between 2BC and 3A protein. As shown in Fig. Fig.6B6B and Table Table3,3, coexpression of Act-2BCah-HRV14 and Bind-3A induced luciferase activity approximately 20% lower than that observed for Act-2BCwt. The decrease in induction of luciferase activity also was detected when these proteins were expressed in the opposite orientation, e.g., Act-3A and Bind-2BCah-HRV14, although in this case, the effect was only ~10%. The 2BCah-HRV14 fusion proteins containing the single amino acid change L28F manifested significantly increased interaction with 3A protein (Fig. (Fig.6B6B and Table Table3).3). Further correlation of 2BC-3A interaction with virus growth was seen when the 2BC-F28L protein, with only a single point mutation in the 2C amphipathic helix shown previously to severely impair virus replication (31), also showed a reduced interaction between 2BC and 3A (Fig. (Fig.6B6B and Table Table33).

TABLE 3.

Effect of mutations on interaction of 2BC proteins with 3A

Compensating mutations in 2B or 3A proteins affect membrane morphology induced by chimeric PV.

In infected cells, all PV nonstructural proteins can be found associated with newly formed vesicles that constitute the membranous replication complexes. Interactions between the viral proteins may have some effect on the formation and resulting morphology of these complexes. Previously, we showed that the vesicles induced by expression of wild-type PV nonstructural proteins appear identical to those induced during infection of cells with poliovirus (29, 31). However, expression of nonstructural proteins from chimeric genome PV-2Cah-HRV14 induced the formation of membrane structures different from those observed in cells infected with wild-type PV (31). We examined the membrane structures induced by chimeric viruses carrying mutations that compensated for the chimera's poor replication properties. Cells were infected with pseudorevertant viruses R6 (encoding the T35A substitution in 2B [Fig. [Fig.7A7A ]), R9 (encoding the K9R substitution in 3A [Fig. [Fig.7B]),7B]), and the previously described R1 (encoding the L28F substitution in the 2C amphipathic helix [31] [Fig. [Fig.7C]).7C]). Examination of the intracellular membrane morphologies was performed 4, 5.5, and 6 h postinfection by electron microscopy. Clusters of vesicles indistinguishable from those induced by wild-type PV infection were observed in cells infected by R1, R6, and R9 viruses. The R9 virus with a compensating mutation in the 3A protein, which produced plaques slightly smaller than those produced by wild-type PV, also demonstrated smaller-than-wild-type clusters of membrane vesicles, and these appeared somewhat later after infection (6 h). Thus, the improved growth properties exhibited by the pseudorevertant viruses correlate with the ability to induce membrane vesicles that resemble the characteristic poliovirus-like replication complexes.

FIG. 7.

Formation of vesicles visualized by electron microscopy in HeLa cells infected with chimeric virus R6 and harvested at 5.5 h (A), infected with R9 and harvested at 6 h (B), or infected with R1 and harvested at 4 h (C). Arrowheads, vesicle clusters; N, (more ...)

DISCUSSION

In a previous study, we found that a chimeric poliovirus in which the predicted N-terminal amphipathic helix in protein 2C was replaced with the corresponding region from HRV14 produced very small plaques on HeLa cell monolayers and displayed delayed kinetics and low yields of RNA in a single-step growth cycle. Among the small plaques, large plaque variants appeared with relatively high frequency. Sequencing through the region encoding protein 2C identified several mutations that accumulated in genomes with improved growth properties. However, the majority of viruses that emerged with improved growth properties (approximately 60%) contained no reversions within the HRV14 amphipathic helix-encoding sequence or anywhere else in the region encoding 2C. In the present study, we performed sequence analyses on those pseudorevertant viral genomes that revealed nucleotide changes in sequences encoding protein 2B or 3A. The sequence changes in the 2B or 3A protein, as well as previously reported changes in protein 2C (31), were not conserved among the isolates. Thus, there was no common reversion strategy; rather, independent mutations could compensate for the poor replication of the poliovirus chimeric genome encoding the HRV14 amphipathic helix in protein 2C. When representative mutations found in the RNA sequences coding for either the 2B or the 3A protein were reconstructed in the original chimeric genome, they were shown to restore growth to normal or near-normal levels. These data suggested that proteins containing 2B or 3A sequences physically interact with 2C (or its precursors) within the replication complex and that such interaction was perturbed when the N-terminal amphipathic helix in 2C was replaced with the HRV14 sequence but was restored by the compensatory changes in either 2B or 3A. Interestingly, a similar conclusion was reached based on the analysis of chimeric PV genomes in which the hydrophobic region in protein 3A was exchanged for the orthologous element from HRV14. Those chimeric viruses also were viable but demonstrated defects in replication that were improved by secondary mutations in 2B-encoding sequences (34). The postulated interaction between 2C and/or 2BC protein and 3A protein seems quite plausible, since all these proteins are localized together in membranous replication complexes. Moreover, it has been shown previously that when 3A is expressed together with 2BC protein, 3A localizes to a lower-density membrane fraction, characteristic of 2BC-containing membranes, than when expressed alone. These data prompted the suggestion that the ensemble of proteins 2BC and 3A stimulates the formation of membranous vesicles most closely resembling the vesicles induced during poliovirus infection (28). Taken together, the results reported from our and others' laboratories indicate a network of functional interactions among 2B, 2C, and 3A protein sequences likely involving the induced membrane structures that form the PV replication complex.

To test whether the identification of compensating mutations resulted from direct physical interactions among the viral proteins, we utilized a mammalian two-hybrid system to demonstrate specific interactions. We performed analyses of homo- and heteromultimerization of PV proteins 2B, 2C, 2BC, 3A, and 3AB in COS cells. This study demonstrated previously unidentified protein-protein interactions between proteins from the P2 and P3 regions of the poliovirus genome. The yeast two-hybrid system was applied previously to catalogue interactions between the nonstructural proteins within the P2 or P3 regions (5, 42), but interactions between proteins from the P2 and P3 regions were not examined. A mammalian two-hybrid analysis was performed for the closely related coxsackie B3 virus proteins 2B, 2C, and 2BC (6). More recently, protein-protein interactions for nonstructural proteins of porcine teschovirus 1, a representative of a different genus of picornavirus, were studied in a yeast two-hybrid system (43). None of the porcine teschovirus P2 proteins were shown to interact with any P3 proteins tested; in that study, however, analysis was performed only with mutant forms of proteins 2BC, 2C, and 3A, which may have affected the results. Table Table44 shows a summary and comparison of the results obtained in all these studies. The majority of interactions observed in our current study are consistent with those subsets examined by others. Major differences were seen in interactions of 3AB, especially in homodimerization of this protein, which gave a very strong positive signal in the previous yeast two-hybrid analysis (42). The failure to detect such interaction in our experiments may have been due to relatively poor expression of Act-3AB in our studies. On the other hand, evidence for 2C-2C multimerization was not detected by others in two-hybrid analyses, and although signals in the present study were relatively low, they were reproducibly observed. 2C homodimerization has been demonstrated previously by glutathione S-transferase pulldown assay (5); multimerization was proposed on the basis of genetic studies of resistance to guanidine-HCl determined by 2C (33); and 2C and 2B clusters were visualized by immunoelectron microscopy of 2B and 2C protein sequences on isolated vesicular membranes of the replication complex (12). A limitation of the two-hybrid methodology for identifying protein interactions, regardless of whether yeast or mammalian cell systems are utilized, is the probability of missing weak interactions, which may be extremely important, especially in regulatory pathways (41).

TABLE 4.

Comparison of picornavirus P2 and P3 protein-protein interactions observed in different studies

Analysis of the effect of replacement of the PV N-terminal amphipathic helix on the demonstrated interactions of 2C-containing proteins revealed an apparent increase in chimeric 2C dimerization. This homodimerization was restored to wild-type levels of binding by a single point mutation at residue 28 (L28F) within the HRV14 sequence, which we had shown previously to also restore the wild-type replication phenotype. In addition, the interactions between PV 3A with the chimeric 2BC protein containing the amphipathic helix from HRV14, or with the PV 2BC protein harboring the single F28L mutation in the 2C sequence, were both reproducibly decreased compared to 3A interactions with the wild-type PV 2BC protein. Again, reversion at 2C-L28F in the chimeric 2BC protein, which rescued the growth of chimeric virus, significantly increased the 2BC-3A interactions. It has also been reported that mutations in protein 2C of HRV16 were sufficient for adaptation of this virus to grow in mouse cells. Interestingly, some of these mutations were shown to affect the dimerization of 2BC protein, as measured by yeast two-hybrid analysis (15).

It remains unclear whether amino acid residues in protein 3A that are changed in viruses with improved growth properties participate directly in interactions with the N-terminal amphipathic helix in protein 2C or if the effect of these mutations is indirect. The amino acid substitutions found in pseudorevertant viruses generally represent conservative changes, and they are sometimes found in corresponding positions of other picornaviruses; however, they are not specific for HRV14. The structure of the soluble domain of the PV protein 3A has been determined by nuclear magnetic resonance spectroscopy (27). The compensating mutation that we found in virus R9 (K9R) is located in the unstructured N-terminal portion of 3A. Other mutations in this region affect 3A's ability to inhibit ER-to-Golgi traffic (7) and 3AB's stimulation of 3CD protease activity (20). The amino acid substitution found in R10 (K39E) is in a solvent-exposed region shown to participate in homodimerization of 3A in solution. Compensating mutation S48R in R11 is located at the junction between a central structured region and an unstructured region linking to the membrane-binding domain. Thus, it is difficult to predict how these mutations would affect the structure of 3A.

For enterovirus 2B proteins, two hydrophobic regions have been identified (3, 39, 40). One of these regions (PV 2B residues 32 to 55) is predicted to form a partially amphipathic cationic helix; the second region (PV 2B residues 61 to 81) is thought to be a transmembrane domain. Mutations that alter either the amphipathic character of the first domain or the hydrophobicity of the second domain have been shown to interfere with the ability of 2B to increase membrane permeability and with viral RNA replication (2, 3, 37, 38, 40). Two of the three mutations in the 2B sequence described in this study are located in the first hydrophobic region; each causes substitution of a polar amino acid by a more hydrophobic one. More detailed structural information will be necessary to understand the effects of the described mutations on the architecture of poliovirus RNA replication complexes.

Although replication of all positive-strand RNA viruses depends upon the reorganization of host intracellular membranes, different viruses utilize different organelle membranes and generate different morphological structures upon which replication complexes assemble. It has been shown that the N-terminal region of nodavirus flock house virus protein A, which normally targets the protein to mitochondrial membranes (18), can be readily replaced by viral or cellular ER-targeting sequences and thus establish replication complexes of greatly differing morphologies without affecting the ability of the virus to replicate. It is not known whether or how the specific morphology of membrane structures induced by PV proteins affects their function in viral RNA replication. In the studies reported here and in our previous publication, the chimeric viruses that generated functional viral replication complexes also formed vesicles with the morphological characteristics of those induced by wild-type PV proteins. Nonfunctional or viable but defective replication complexes (e.g., those formed by PV-2Cah-HRV14) appeared altered in their morphological aspects but exhibited the typical PV vesicle clusters when function was restored by mutations in the same or other proteins that restored replication activity. Thus, for the replication-defective viruses analyzed in these studies, we observed a positive correlation between the induced membrane structures and the capacity to replicate the virus genome.

Acknowledgments

This research was supported in part by the Intramural Research Program of the NIH, NIAID.

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