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.     

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.     

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.     

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.     

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.     

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.     

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.     

Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of p2 proteins

The replication proteins encoded in the P2 region of the poliovirus genome induce extensive rearrangement of cellular membranes into vesicles and are a required component of viral RNA replication complexes. To identify distinct viral protein(s) from the P2 region of the genome that were required to form functional RNA replication complexes, the P2 proteins were expressed in addition to P3 in HeLa S10 translation-RNA replication reactions. Membrane-associated preinitiation replication complexes were isolated from these reactions and used to measure negative-strand synthesis. The formation of replication complexes capable of initiating negative-strand synthesis was observed when either P23 or when P2 and P3 were expressed in the HeLa S10 translation-replication reactions. The amount of negative-strand RNA synthesized with P2 and P3 was approximately 50% of that observed with P23. Negative-strand synthesis was not observed when the processed forms of the P2 proteins (e.g., 2A, 2B, 2C, 2AB, and 2BC) were used in various combinations in place of P2. In contrast, the expression of 2A and 2BCP3 supported negative-strand synthesis at the same level observed with P23. Therefore, functional replication complexes were formed in reaction mixtures that contained either 2A and 2BCP3 or P2 and P3. Genetic complementation analysis of P23 RNA that contained a lethal mutation in 2C confirmed these results. The expression of 2BCP3 in trans restored the replication of P23-2C(P131N) RNA to wild-type levels. The expression of P2 and P3 also complemented the replication of this mutant RNA, although very inefficiently. Complementation was not observed in reactions that contained P2 alone, 2BC, or 2C. Based on these results, we propose that RNA replication complexes are initially formed with the primary cleavage products of P23 (i.e., P2 and P3 or 2A and 2BCP3), and that 2A and 2BCP3 are preferentially used in this process.     

Poliovirus cis-acting replication element-dependent VPg Uridylylation lowers the Km of the initiating nucleoside triphosphate for viral RNA replication

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5′ terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5′ end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpU_OH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpU_OH-independent manner, CRE-dependent VPgpUpU_OH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the K_m of UTP required for viral RNA replication and that CRE-dependent VPgpUpU_OH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.     

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.     

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.     

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.     

Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.     

Replication of poliovirus RNA with complete internal ribosome entry site deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.     

Mutational analysis of the rubella virus nonstructural polyprotein and its cleavage products in virus replication and RNA synthesis

Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.     

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.     

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.