A tandem repeat gene in a picornavirus.

Three closely related genes for the small genome-linked protein (VPg) of picornaviruses have been identified by sequence analysis as a tandem repeat in the genome of Foot and Mouth Disease Virus (FMDV), strain O1K. This unusual structure was also found in the genome of strain C1O, belonging to a different FMDV serotype. Predicted biochemical properties of the three VPg gene products are in excellent agreement with the data from protein analysis of a heterogeneous VPg population from a third FMDV serotype, strain A10 (1). Taken together, these data indicate that the VPgs from all three genes function equally well in vivo. This is the first report of a tandem repeat gene in a viral genome.     

Sobemovirus RNA linked to VPg over a threonine residue

Positive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5' end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg.     

Genetic variation of the poliovirus genome with two VPg coding units.

Amongst the picornaviruses, poliovirus encodes a single copy of the genome-linked protein, VPg wheras foot-and-mouth disease virus uniquely encodes three copies of VPg. We have previously shown that a genetically engineered poliovirus genome containing two tandemly arranged VPgs is quasi-infectious (qi) that, upon genome replication, inadvertently deleted one complete VPg sequence. Using two genetically marked viral genomes with two VPg sequences, we now provide evidence that this deletion occurs via homologous recombination. The mechanism was abrogated when the second VPg was engineered such that its nucleotide sequence differed from that of the first VPg sequence by 36%. Such genomes also expressed a qi phenotype, but progeny viruses resulted from (i) random deletions yielding single VPg coding sequences of varying length lacking the Q*G cleavage site between the VPgs and (ii) mutations in the AKVQ*G cleavage sites between the VPgs at either the P4, P1 or P1' position. These variants present a unique genetic system defining the cleavage signals recognized in 3Cpro-catalyzed proteolysis. We propose a recognition event in the cis cleavages of the polyprotein P2-P3 region, and we present a hypothesis why the poliovirus genome does not tolerate two tandemly arranged VPg sequences.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

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.     

Mutational analysis of the genome-linked protein VPg of poliovirus.

Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

Insights into RNA virus mutant spectrum and lethal mutagenesis events: replicative interference and complementation by multiple point mutants

RNA virus behavior can be influenced by interactions among viral genomes and their expression products within the mutant spectra of replicating viral quasispecies. Here, we report the extent of interference of specific capsid and polymerase mutants of foot-and-mouth disease virus (FMDV) on replication of wild-type (wt) RNA. The capsid and polymerase mutants chosen for this analysis had been characterized biochemically and structurally. Upon co-electroporation of BHK-21 cells with wt RNA and a tenfold excess of mutant RNA, some mutants displayed strong interference (<10% of progeny production by wt RNA alone), while other mutants did not show detectable interference. The capacity to interfere required an excess of mutant RNA and was associated with intracellular replication, irrespective of the formation of infectious particles by the mutant virus. The extent of interference did not correlate with the known types and number of interactions involving the amino acid residue affected in each mutant. Synergistic interference was observed upon co-electroporation of wt RNA and mixtures of capsid and polymerase mutants. Interference was specific, in that the mutants did not affect expression of encephalomyocarditis virus RNA, and that a two nucleotide insertion mutant of FMDV expressing a truncated polymerase did not exert any detectable interference. The results support the lethal defection model for viral extinction by enhanced mutagenesis, and provide further evidence that the population behavior of highly variable viruses can be influenced strongly by the composition of the quasispecies mutant spectrum as a whole.     

Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg

The replication of the foot-and-mouth disease virus (FMDV) genome is critically dependent upon the activity of a virally encoded RNA-dependent RNA polymerase (RdRp). In this study, four mutant RdRps of FMDV were isolated from viral quasi-species treated with ribavirin, of which two were single mutants (L123F and T381A) and two were double mutants (T291I/T381I and L123F/F244L). The mutant proteins were expressed in Escherichia coli and purified by His-bind resin chromatography. In combination with real-time RT-PCR, an in vitro RNA replication system that uses genome RNA/VPg as template-primers was used to determine polymerase activity. Mutant L123F exhibited a 0.6-fold decrease (p < 0.001) in polymerase activity relative to wild-type RdRp, whereas the activity of L123F/F244L and T381A was undetectable. Surprisingly, the activity of T291I/T381I yielded a 0.7-fold increase (p < 0.001) as compared to wild-type. In order to study the structure-function relationship of RdRp, all structures of the RdRp-RNA template-primer complex were obtained through homology modeling and molecular docking. The VPg1 orientation in the RdRp-VPg1 complexes was determined and analyzed with mathematical methods. Our results reveal that the orientation of VPg after binding to the polymerase determines the FMDV RdRp catalytic activity, which provides a basis for the rational design of novel antiviral agents.     

Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.     

Identification of Human Astrovirus Genome-Linked Protein (VPg) Essential for Virus Infectivity

Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family Astroviridae has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of Caliciviridae has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.     

Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis.

By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.     

Structural flexibility allows the functional diversity of potyvirus genome-linked protein VPg

Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel β-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility.     

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.     

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis.

A molecular genetic analysis has been combined with an in vitro biochemical approach to define the functional interactions required for nucleotidyl protein formation during poliovirus RNA synthesis. A site-directed lesion into the hydrophobic domain of a viral membrane protein produced a mutant virus that is defective in RNA synthesis at 39 degrees C. The phenotypic expression of this lesion affects initiation of RNA synthesis, in vitro uridylylation of the genome-linked protein (VPg), and the in vivo synthesis of plus-strand viral RNAs. Our results support a model that employs a viral membrane protein as carrier for VPg in the initiation of plus-strand RNA synthesis. Our data also suggest that a separate mechanism could be used in the initiation of minus-strand RNA synthesis, thereby providing a means for strand-specific regulation of picornavirus RNA 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.     

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.     

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.