An electron microscope study of the proteins attached to polio virus RNA and its replicative form (RF).

A recently described method (Wu, M. and Davidson, N. (1978), Nucleic Acids Research 5, in press) for visualizing proteins attached to nucleic acids in the electron microscope has been applied to study proteins attached to poliovirion RNA and to the viral double-stranded intracellular RF form. A protein is found at the 5' end of the plus strand virion RNA, and protein components are found at both ends of the duplex RF. In the RF as usually extracted, there is frequently a larger or compound protein aggregate at the end which contains the 3' end of the plus strand and the 5' end of the minus strand. Banding in CsCl-guanidinium hydrochloride in the presence of sarkosyl causes dissociation of some components of this aggregate, leaving both ends labeled with the covalently bound VPg. These results confirm and extend previous biochemical studies of proteins bound to poliovirion RNA and to the RF form.     

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.     

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.     

Novel, structure-based mechanism for uridylylation of the genome-linked peptide (VPg) of picornaviruses

The VPg peptide, which is found in poliovirus infected cells either covalently bound to the 5'-end of both plus and minus strand viral RNA, or in a uridylylated free form, is essential for picornavirus replication. Combining experimental structure and mutation results with molecular modeling suggests a new mechanism for VPg uridylylation, which assigns an additional function, that of scaffold, to the polymerase. The polarity of the NMR structure of VPg is complementary to the binding site on the surface of poliovirus polymerase determined previously by mutagenesis. Docking VPg at this position places the reactive tyrosinate close to the 5'-end of Poly(A)7 RNA when this is bound with its 3'-end in the active site of the polymerase. The triphosphate tail of a UTP moiety, base paired with the 5'-end of the RNA, projects back over the Tyr3-OH and is held in position by conserved positively charged side-chains of VPg. Other conserved residues mediate binding to the polymerase surface and serve as ligands for metal ion catalyzed transphosphorylation. Additional viral proteins or a second polymerase molecule may aid in stabilizing the components of the reaction. In the model complex, VPg can direct its own uridylylation before entering the polymerase active site.     

Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.     

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.     

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.     

Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show approximately 2.6-fold faster association for eIFiso4F.VPg as compared with eIF4F.VPg. The dissociation rate was approximately 2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F.VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.     

Localization of a potyvirus and the viral genome-linked protein in wild potato leaves at an early stage of systemic infection

The upper noninoculated 'sink' leaves of the wild potato species, Solanum commersonii, were studied for distribution of Potato virus A (PVA) at an early stage of systemic infection. Viral RNA was detected by in situ hybridization, and five viral proteins were localized using immunohistochemical staining in leaf sections. Initial systemic infection foci were found at the vicinity of major and minor veins. In these infection foci, the viral coat protein, cylindrical inclusion protein, and helper component-proteinase colocalized with viral RNA in parenchyma and mesophyll cells, but none of these were detected in companion cells (CC). In contrast, VPg, which is the N-proximal half of the NIa protein (separated from the C-terminal proteinase domain, NIapro, by an autocatalytic cleavage) and acts as a viral genome-linked protein, was detected in CC in the infection foci, but only at an early stage of virus unloading. Outside the infection foci, conspicuous signals for VPg were readily and exclusively detected in CC of many veins in all vein classes in the absence of signals for NIapro, other viral proteins, and viral RNA. Taken together, our data indicate that both major and minor veins may unload PVA in the sink leaves of potato. The data suggest that VPg is translocated from inoculated source leaves to the sink leaves, where it accumulates in CC at an early stage of systemic infection. These findings suggest that VPg may be a 'phloem protein' that specifically acts in CC in the sink leaves to facilitate virus unloading.     

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.     

A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNa polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNa replication complex.     

The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg. The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA. This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors. The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis. VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates. To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES. VPg inhibited translation of all reporter RNAs in a dose-dependent manner. Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral 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.     

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.     

Tyrosine 3 of poliovirus terminal peptide VPg(3B) has an essential function in RNA replication in the context of its precursor protein, 3AB

Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3D(pol), but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3D(pol). Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.     

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.