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

ABSTRACT: 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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC1900252 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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

Liu Ying Y Franco David D Paul Aniko V AV Wimmer Eckard E

Journal of virology 20070314 11

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 infor ...[more]

PMID: 17360746

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Project description:Poliovirus (PV), a model for interactions of picornaviruses with host cells, replicates its genomic RNA in association with cellular membranes. The origin of PV replication membranes has not been determined. Hypotheses about the origin of replication membranes, based largely on localization of viral proteins, include modification of coat protein complex I (COPI) and/or COPII secretory pathway vesicles and subversion of autophagic membranes. Here, we use an antibody against double-stranded RNA (dsRNA) to identify replication complexes by detection of dsRNA replication intermediates. dsRNA signal is dependent on virus genome replication and colocalizes with the viral integral membrane protein 3A, which is part of the RNA replication complex. We show that early in infection, dsRNA does not colocalize with a marker for autophagic vesicles, making it unlikely that autophagosomes contribute to the generation of PV RNA replication membranes. We also find that dsRNA does not colocalize with a marker of the COPII coat, Sec31, and, in fact, we demonstrate proteasome-dependent loss of full-length Sec31 during PV infection. These data indicate that COPII vesicles are an unlikely source of PV replication membranes. We show that the Golgi resident G-protein Arf1 and its associated guanine nucleotide exchange factor (GEF), GBF1, transiently colocalize with dsRNA early in infection. In uninfected cells, Arf1 nucleates COPI coat formation, although during infection the COPI coat itself does not colocalize with dsRNA. Phosphatidylinositol-4-phosphate, which is associated with enterovirus-induced vesicles, tightly colocalizes with Arf1/GBF1 throughout infection. Our data point to a noncanonical role for some of the COPI-generating machinery in producing unique replication surfaces for PV RNA replication. IMPORTANCE Picornaviruses are a diverse and major cause of human disease, and their genomes replicate in association with intracellular membranes. There are multiple hypotheses to explain the nature and origin of these membranes, and a complete understanding of the host requirements for membrane rearrangement would provide novel drug targets essential for viral genome replication. Here, we study the model picornavirus, poliovirus, and show that some, but not all, components of the cellular machinery required for retrograde traffic from the Golgi apparatus to the endoplasmic reticulum are transiently present at the sites of viral RNA replication. We also show that the full-length Sec31 protein, which has been suggested to be present on PV RNA replication membranes, is lost during infection in a proteasome-dependent manner. This study helps to reconcile multiple hypotheses about the origin of poliovirus replication membranes and points to known host cell protein complexes that would make likely drug targets to inhibit picornavirus infections.

Dataset Information

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

Publications

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

Similar Datasets

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