Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.     

Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis

The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3' poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.     

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.     

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.     

Poly(rC) binding proteins mediate poliovirus mRNA stability

The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA.     

3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation

The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.     

Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

A new role for ns polyprotein cleavage in Sindbis virus replication

One of the distinguishing features of the alphaviruses is a sequential processing of the nonstructural polyproteins P1234 and P123. In the early stages of the infection, the complex of P123+nsP4 forms the primary replication complexes (RCs) that function in negative-strand RNA synthesis. The following processing steps make nsP1+P23+nsP4, and later nsP1+nsP2+nsP3+nsP4. The latter mature complex is active in positive-strand RNA synthesis but can no longer produce negative strands. However, the regulation of negative- and positive-strand RNA synthesis apparently is not the only function of ns polyprotein processing. In this study, we developed Sindbis virus mutants that were incapable of either P23 or P123 cleavage. Both mutants replicated in BHK-21 cells to levels comparable to those of the cleavage-competent virus. They continuously produced negative-strand RNA, but its synthesis was blocked by the translation inhibitor cycloheximide. Thus, after negative-strand synthesis, the ns proteins appeared to irreversibly change conformation and formed mature RCs, in spite of the lack of ns polyprotein cleavage. However, in the cells having no defects in alpha/beta interferon (IFN-alpha/beta) production and signaling, the cleavage-deficient viruses induced a high level of type I IFN and were incapable of causing the spread of infection. Moreover, the P123-cleavage-deficient virus was readily eliminated, even from the already infected cells. We speculate that this inability of the viruses with unprocessed polyprotein to productively replicate in the IFN-competent cells and in the cells of mosquito origin was an additional, important factor in ns polyprotein cleavage development. In the case of the Old World alphaviruses, it leads to the release of nsP2 protein, which plays a critical role in inhibiting the cellular antiviral response.     

Conversion of VPg into VPgpUpUOH before and during Poliovirus Negative-Strand RNA Synthesis

There are two protein primers involved in picornavirus RNA replication, VPg, the viral protein of the genome, and VPgpUpU_OH. A cis-acting replication element (CRE) within the open reading frame of poliovirus (PV) RNA allows the viral RNA-dependent RNA polymerase 3D^Pol to catalyze the conversion of VPg into VPgpUpU_OH. In this study, we used preinitiation RNA replication complexes (PIRCs) to determine when CRE-dependent VPg uridylylation occurs relative to the sequential synthesis of negative- and positive-strand RNA. Guanidine HCl (2 mM), a reversible inhibitor of PV 2C^ATPase, prevented CRE-dependent VPgpUpU_OH synthesis and the initiation of negative-strand RNA synthesis. VPgpUpU_OH and nascent negative-strand RNA molecules were synthesized coincident in time following the removal of guanidine, consistent with PV RNA functioning simultaneously as a template for CRE-dependent VPgpUpU_OH synthesis and negative-strand RNA synthesis. The amounts of [³²P]UMP incorporated into VPgpUpU_OH and negative-strand RNA products indicated that 100 to 400 VPgpUpU_OH molecules were made coincident in time with each negative-strand RNA. 3′-dCTP inhibited the elongation of nascent negative-strand RNAs without affecting CRE-dependent VPg uridylylation. A 3′ nontranslated region mutation which inhibited negative-strand RNA synthesis did not inhibit CRE-dependent VPg uridylylation. Together, the data implicate 2C^ATPase in the mechanisms whereby PV RNA functions as a template for reiterative CRE-dependent VPg uridylylation before and during negative-strand RNA synthesis.A common feature of positive-strand RNA viruses is the asymmetric replication of viral RNA. Poliovirus (PV) RNA replication has been studied extensively; however, it remains to be determined exactly how the synthesis of negative-strand RNA and that of positive-strand RNA are mechanistically distinct, culminating in the synthesis of greater amounts of positive-strand than negative-strand RNA (2). A cis-acting replication element (CRE) within the 2C open reading frame of PV RNA functions as a template for the conversion of the viral protein of the genome (VPg) into VPgpUpU_OH (24, 26, 37). 3D polymerase (3D^Pol), in concert with other viral proteins, catalyzes the conversion of VPg into VPgpUpU_OH on CRE RNA templates (22). It remains to be determined whether the tyrosine hydroxyl of VPg (14, 20, 21), the 3′ hydroxyl of VPgpUpU_OH (22, 23, 43), or both (38) are used to prime negative-strand RNA synthesis. It would be informative to know whether VPg is converted into VPgpUpU_OH before, during, and/or after the initiation of viral negative-strand RNA synthesis. Conversion of VPg into VPgpUpU_OH before the initiation of negative-strand RNA synthesis would be consistent with the possibility that it primes the initiation of negative-strand RNA synthesis. Conversely, if VPg were not converted into VPgpUpU_OH until after the initiation of negative-strand RNA synthesis, VPgpUpU_OH could not possibly participate in the initiation of negative-strand RNA synthesis. Also, because multiple copies of VPgpUpU_OH are necessary to prime reiterative initiation of positive-strand RNA synthesis (35), VPg is most likely converted into abundant amounts of VPgpUpU_OH before the initiation of positive-strand RNA synthesis.PV preinitiation RNA replication complexes (PIRCs) were used in this study to examine when VPg is converted into VPgpUpU_OH. PIRCs assemble and accumulate when PV mRNA is translated in reaction mixtures containing cytoplasmic extracts from uninfected HeLa cells and 2 mM guanidine HCl, a reversible inhibitor of viral RNA replication (5). The viral replication proteins expressed from the viral mRNA interact with lipid membranes in the cytoplasmic extracts to form RNA replication complexes (RCs) similar to those in infected cells (12). PIRCs convert VPg into VPgpUpU_OH and initiate viral RNA replication when they are isolated from reaction mixtures containing guanidine and resuspended in reaction mixtures lacking guanidine (6, 19). Guanidine HCl functions as a reversible inhibitor of PV RNA replication, both in cells (11) and in cell-free translation-replication reactions (6). In cells, PV RNA RCs fail to immediately initiate RNA replication following the removal of guanidine HCl (11). Rather, PV RCs formed in the presence of guanidine in cells appear to be translocated to a region of the cytoplasm where the RCs and their contents may be recycled and/or destroyed (11), possibly by autophagic vesicles (17). Recycling and/or destruction of RCs by autophagic vesicles would preclude their function upon the removal of guanidine. PIRCs, which form in the presence of guanidine during the translation of PV mRNA in cytoplasmic extracts of HeLa cells, immediately initiate both negative-strand RNA synthesis and CRE-dependent VPg uridylylation upon the removal of guanidine (6, 19). Viral RNA replication and VPgpUpU_OH synthesis are monitored by the incorporation of radiolabeled UTP (19-21). It is important to note that RNA replication in the context of PIRCs is artificial in that the PIRCs are stalled with guanidine and purified and then the guanidine block is removed. Despite this artificiality, the mechanisms of RNA replication within PIRCs appear to reliably represent the mechanisms of RNA replication in cells. There are several advantageous features of the PIRC experimental system: viral RNA replication is synchronous and sequential, with negative-strand RNA being made before positive-strand RNA (6); viral RNA replication is asymmetric, with an excess of positive-strand RNA being made from each negative-strand template; VPg is converted into VPgpUpU_OH in a CRE-dependent manner (20, 21); and the reaction conditions, including nucleoside triphosphate concentrations, are easily manipulated (38). Importantly, PIRCs contain all of the viral proteins associated with RNA replication and RNA replication by PIRCs faithfully mimics the asymmetric replication of PV RNA observed in cells.PV protein 2C, the target of guanidine HCl (30), is a critical but poorly understood component of PIRCs and RNA RCs in cells. PV protein 2C has an NH-terminal amphipathic helix which interacts with cellular membranes (40), a central ATPase domain where guanidine-resistant and guanidine-dependent mutations arise (31, 32), a cysteine-rich zinc binding motif (29), and a COOH-terminal RNA binding domain (34) which appears to work in concert with amino acid residues at the NH terminus to bind RNA. 2C^ATPase can oligomerize (1, 41), anchoring viral replication proteins and RNA templates within membranous RCs (4). The ability of guanidine HCl to reversibly inhibit both CRE-dependent VPg uridylylation and negative-strand RNA synthesis implicates 2C^ATPase in the mechanisms by which PV RNA functions coordinately as a template for both RNA replication and CRE-dependent VPgpUpU_OH synthesis (19, 21).In this study, we found that VPg was converted into VPgpUpU_OH before and during negative-strand RNA synthesis and that 2C^ATPase activity, in the context of membranous PIRCs, allowed PV RNA to function simultaneously as a template for CRE-dependent VPg uridylylation and as a template for negative-strand RNA synthesis. We discuss how picornaviruses coordinate the synthesis of nucleotidylylated protein primers with other steps of viral RNA replication.     

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions.

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.     

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.     

Strand-specific RNA synthesis defects in a poliovirus with a mutation in protein 3A

Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.     

Identification of determinants involved in initiation of hepatitis C virus RNA synthesis by using intergenotypic replicase chimeras

The 5' nontranslated region (NTR) and the X tail in the 3' NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5' NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5' NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3' end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5' NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.