Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation

The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.     

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.Poliovirus (PV) is the most extensively studied member of the picornavirus family and serves as a paradigm not only for picornaviruses but also for many of the nonretroviral positive strand RNA viruses (74). A schematic of the ∼7,500-nucleotide PV genome is shown in Fig. ​Fig.1A.1A. The 5′ end is linked covalently to a 22-amino-acid peptide termed VPg (virion protein genome linked) that is encoded by the 3B region of the genome. VPg and 3B are therefore used interchangeably. The 3′ end of the genome is terminated by a poly(rA) tail. Upon release of the genome into the host cell cytoplasm, genome translation is initiated by using the internal ribosome entry site. An ∼3,000-amino-acid polyprotein is produced. Complete cleavage of the polyprotein by virus-encoded proteases yields 10 proteins. The polyprotein can be divided further into three smaller polyproteins: P1, P2, and P3. P1 contains capsid proteins: VP0, VP3, and VP1. VP0 undergoes autocatalytic cleavage after genome encapsidation to produce VP4 and VP2 proteins. P2 performs host interaction functions required for robust virus multiplication, for example, shutoff of host cell translation and induction of vesicles employed for genome replication, the so-called replication complexes (RCs). P3 contains proteins that function most directly in genome replication, including the RNA-dependent RNA polymerase. Translation induces RCs, leading to genome replication. Early during infection, replicated genomes are employed as templates for translation, leading to an exponential amplification of RCs and replicated RNA. Ultimately, production of viral proteins ceases and replicated genomes are packaged. The use of RCs provides a barrier to genetic complementation; all proteins must be provided in cis, that is, produced from the RNA that they replicate.Open in a separate windowFIG. 1.PV genome organization and P3 processing pathway. (A) Schematic of the PV genome. The 5′ end of the genome is covalently linked to a peptide (VPg) encoded by the 3B region of the genome. The 3′ end contains a poly(rA) tail. Three cis-acting replication elements are known. oriL is located in 5′ NTR. oriR is located in the 3′ NTR. oriI is located in 2C-coding sequence for PV; the position of this element is virus dependent. oriI is the template for VPg uridylylation. Translation initiation employs an internal ribosome entry site (IRES). The single open reading frame encodes a polyprotein. P1 produces virion structural proteins as indicated. P2 produces proteins thought to participate in virus-host interactions required for genome replication. P3 produces proteins thought to participate directly in genome replication. Polyprotein processing is mediated by protease activity residing in 2A, 3C, and/or 3CD proteins. (B) Processing of the P3 precursor occurs by two independent pathways (60). There are major (I) and minor (II) pathways. In pathway I, processing between 3B and 3C yields 3AB and 3CD. In pathway II, processing between 3A and 3B yields 3A and 3BCD. 3BCD processing yields 3BC and 3D; 3BC processing yields 3B and 3C. Pathway II is proposed to function in genome replication and is not perturbed in the GG mutant.In addition to P3 proteins, genome replication requires three cis-acting replication elements (CREs): a cloverleaf structure located in the 5′ nontranslated region (NTR), termed oriL (left) (1, 5); a stem-loop structure located in 2C-coding sequence, termed oriI (internal) (30, 61); and a pseudoknot structure located in the 3′ NTR, termed oriR (right) (1, 40). The first step of genome replication is diuridylylation of VPg or a VPg-containing protein primer (62, 74). This reaction is templated by oriI but also requires oriL in a cell-free reaction and is catalyzed by the viral RNA-dependent RNA polymerase 3Dpol (4, 5, 11, 30, 61). In addition to the four terminal P3 cleavage products (3A, 3B, 3C, and 3D proteins) and the uncleaved P3 polyprotein, several “intermediates” are observed in infected cells (3AB, 3CD, and 3BCD proteins) (Fig. ​(Fig.1B)1B) (43, 57, 73). The major P3 cleavage pathway (I) produces 3AB and 3CD proteins; the minor P3 cleavage pathway (II) produces 3A and 3BCD proteins (Fig. ​(Fig.1B)1B) (60). In some cases, the intermediates have unique activities, specificities, and/or functions relative to their corresponding terminal cleavage products.Over the past 8 years much has been learned about oriI-templated VPg uridylylation in vitro for a variety of picornaviruses (28, 49, 53, 77, 92). However, it is still unclear whether or not VPg, 3BC(D), or 3AB is used in vivo to initiate genome replication. The VPg peptide can be uridylylated in vitro (62); however, VPg-pUpU does not chase efficiently into full-length RNA (81). 3BC(D) is uridylylated more efficiently than VPg in vitro, leading to the possibility that this precursor could be used in vivo (60). To date, 3AB has been uridylylated in vitro only in the presence of Mn²⁺ (66). In order to begin to probe the origin of VPg that is linked to picornaviral RNA, we created a PV mutant in which the cleavage site between 3B and 3C was changed from Gln-Gly to Gly-Gly (60). We refer to this mutant as GG. The GG mutation should be lethal for genome replication if use of the processed VPg peptide is absolutely required for genome replication. For the GG mutant, products of the major P3 cleavage pathway were no longer 3AB and 3CD but were now 3ABC and 3D instead. The kinetics of genome replication were reduced for the GG mutant relative to those for the wild type (WT). Surprisingly, the yield of replicated GG RNA was within an order of magnitude of that observed for WT RNA. Replicated GG RNA was then linked covalently to 3BC instead of VPg, as observed for WT PV. In spite of the substantial yield of replicated RNA, infectious virus was not recovered.We have performed a molecular characterization of the GG mutant. GG PV RNA is quasi-infectious. The low yield of virus recovery relative to replicated RNA reflects a block at a step in the PV multiplication cycle positioned after genome replication but prior to virus assembly. The existence of this step in the PV life cycle was suggested previously by Baltimore (8). Surprisingly, none of the defects associated with GG PV could be attributed to the absence of 3CD protease activity, suggesting that precursors larger than 3CD may be the primary proteases employed in vivo. All of the observed defects in GG PV multiplication were ameliorated in a pseudorevertant in which the 3B-3C cleavage site was changed from Gly-Gly to Glu-Gly. This mutant is referred to as EG. Molecular characterization of EG PV revealed for the first time a trans-complementable function for 3CD in genome replication. This observation supports a role for 3CD at a step preceding genome replication within RCs, perhaps RC formation. Our studies of EG PV confirmed the existence of a step between genome replication and virus assembly that requires 3CD and/or 3AB, thus providing compelling evidence for genome replication and genome encapsidation as distinct steps in the multiplication cycle. This study highlights the utility of polyprotein cleavage site mutants for evaluation of the viral multiplication cycle.     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.     

Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB.

Genome replication of poliovirus, as yet unsolved, involves numerous viral polypeptides that arise from proteolysis of the viral polyprotein. One of these proteins is 3AB, an RNA-binding protein with multiple functions, that serves also as the precursor for the genome-linked protein VPg (= 3B). Eight clustered charged amino acid-to-alanine mutants in the 3AB coding region of poliovirus were constructed and analyzed, together with three additional single-amino acid exchange mutants in VPg, for viral phenotypes. All mutants expressed severe inhibition in RNA synthesis, but none were temperature sensitive (ts). The 3AB polypeptides of mutants with a lethal phenotype were overexpressed in Escherichia coli, purified to near homogeneity, and studied with respect to four functions: (1) ribonucleoprotein complex formation with 3CDpro and the 5'-terminal cloverleaf of the poliovirus genome; (2) binding to the genomic and negative-sense RNA; (3) stimulation of 3CDpro cleavage; and (4) stimulation of RNA polymerase activity of 3Dpol. The results have allowed mapping of domains important for RNA binding and the formation of certain protein-protein complexes, and correlation of these processes with essential steps in viral genome replication.     

Characterization of protein-protein interactions critical for poliovirus replication: analysis of 3AB and VPg binding to the RNA-dependent RNA polymerase

Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 microM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.     

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.     

Genetic evidence for an interaction between a picornaviral cis-acting RNA replication element and 3CD protein

Internally located, cis-acting RNA replication elements, termed cres, are essential for replication of the genomes of picornaviruses such as human rhinovirus 14 (HRV-14) and poliovirus because they template uridylylation of the protein primer, VPg, by the polymerase 3D(pol). These cres form stem-loop structures sharing a common loop motif, and the HRV-14 cre can substitute functionally for the poliovirus cre in both uridylylation in vitro and RNA replication in vivo. We show, however, that the poliovirus cre is unable to support HRV-14 RNA replication. This lack of complementation maps to the stem of the poliovirus cre and was reversed by single nucleotide substitutions in the stem as well as the base of the loop. Replication-competent, revertant viruses rescued from dicistronic HRV-14 RNAs containing the poliovirus cre, or a chimeric cre containing the poliovirus stem, contained adaptive amino acid substitutions. These mapped to the surface of both the polymerase 3D(pol), at the tip of the "thumb" domain, and the protease 3C(pro), on the side opposing the active site and near the end of an extended strand segment implicated previously in RNA binding. These mutations substantially enhanced replication competence when introduced into HRV-14 RNAs containing the poliovirus cre, and they were additive in their effects. The data support a model in which 3CD or its derivatives 3C(pro) and 3D(pol) interact directly with the stem of the cre during uridylylation of VPg.     

Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection

Poliovirus (PV) modifies membrane-trafficking machinery in host cells for its viral RNA replication. To date, ARF1, ACBD3, BIG1/BIG2, GBF1, RTN3, and PI4KB have been identified as host factors of enterovirus (EV), including PV, involved in membrane traffic. In this study, we performed small interfering RNA (siRNA) screening targeting membrane-trafficking genes for host factors required for PV replication. We identified valosin-containing protein (VCP/p97) as a host factor of PV replication required after viral protein synthesis, and its ATPase activity was essential for PV replication. VCP colocalized with viral proteins 2BC/2C and 3AB/3B in PV-infected cells and showed an interaction with 2BC and 3AB but not with 2C and 3A. Knockdown of VCP did not suppress the replication of coxsackievirus B3 or Aichi virus. A VCP-knockdown-resistant PV mutant had an A4881G (a mutation of E253G in 2C) mutation, which is known as a determinant of a secretion inhibition-negative phenotype. However, knockdown of VCP did not affect the inhibition of cellular protein secretion caused by overexpression of each individual viral protein. These results suggested that VCP is a host factor required for viral RNA replication of PV among membrane-trafficking proteins and provides a novel link between cellular protein secretion and viral RNA replication.     

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.     

Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.     

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.     

Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C(pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol) at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D(pol) G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D(pol) makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.     

Enterovirus 71 VPg Uridylation Uses a Two-Molecular Mechanism of 3D Polymerase

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D^pol) and the transfer of UMP by 3D^pol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D^pol) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D^pol by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D^pol, which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D^pol, which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D^pol. Mutation of a polymerase active site in 3D^pol and Site-311 in 3D^pol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro. These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D^pol during EV71 VPg uridylylation, such that one 3D^pol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D^pol, which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.     

Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg

Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn(2+) than Mg(2+) and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [alpha-(32)P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses.     

Picornavirus genome replication: assembly and organization of the VPg uridylylation ribonucleoprotein (initiation) complex

All picornaviruses have a protein, VPg, covalently linked to the 5'-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication.     

Factors required for the Uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.     

cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.     

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.