Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

Takegami, Tsutomu; Semler, Bert L.; Anderson, Carl W.; Wimmer, Eckard

doi:10.1016/0042-6822(83)90316-1

Cited by 136 publications

(140 citation statements)

References 24 publications

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“…These antibodies detected no free VPg in infected cells but specifically immunoprecipitated three larger polypeptides with VPg determinants from infected cell lysates, of which the smallest one had a molecular weight of 12,000 (pre-VPg 3 or P3-9) (7)(8)(9). In poliovirus replicase reactions, anti-VPg antibody specifically inhibited initiation of viral RNA synthesis and immunoprecipitated polynucleotides synthesized by the viral replicase (10).…”

mentioning

confidence: 99%

“…The intracellular forms of VPg and their possible role in viral replication have been studied previously using antibodies made to chemically synthesized segments of VPg (7)(8)(9). These antibodies detected no free VPg in infected cells but specifically immunoprecipitated three larger polypeptides with VPg determinants from infected cell lysates, of which the smallest one had a molecular weight of 12,000 (pre-VPg 3 or P3-9) (7)(8)(9).…”

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confidence: 99%

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Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.

Crawford¹,

Baltimore²

1983

Proc. Natl. Acad. Sci. U.S.A.

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VPg is a virus-encoded protein covalently attached to the 5' end of poliovirus virion RNA. We have used antibody prepared against chemically synthesized VPg to detect two forms of VPg in infected cells. Both forms were specifically immunoprecipitated from lysates of infected cells labeled with [3H]-leucine. One appears to be unmodified VPg because it had the same electrophoretic mobility as synthetic VPg. The other had a larger apparent molecular weight than VPg and could be labeled in vivo with 32p,. Its structure is VPg-pUpU, the UMP dinucleotide being attached to VPg via a phosphodiester bond to tyrosine, the third amino acid from the NH2 terminus of VPg. This structure is identical to that found at the 5' end of virion and minus-strand RNA.A 22-amino acid protein, VPg, is attached to the 5' end of the single-stranded RNA genome of poliovirus (1-3). VPg is linked to viral RNA by a tyrosine-phosphate bond in the structure, VPg(Tyr-04)-pU-U-A-A . . (4,5). VPg is also found at the 5' end of intracellular plus and minus strand RNA, and on nascent plus strands; these observations suggest that VPg may act as a primer of poliovirus RNA synthesis (2,3,6).The intracellular forms of VPg and their possible role in viral replication have been studied previously using antibodies made to chemically synthesized segments of VPg (7-9). These antibodies detected no free VPg in infected cells but specifically immunoprecipitated three larger polypeptides with VPg determinants from infected cell lysates, of which the smallest one had a molecular weight of 12,000 (pre-VPg 3 or P3-9) (7-9). In poliovirus replicase reactions, anti-VPg antibody specifically inhibited initiation of viral RNA synthesis and immunoprecipitated polynucleotides synthesized by the viral replicase (10). These results imply that protein with VPg determinants is attached to replicase reaction products and may initiate RNA synthesis. The inability to detect free VPg in infected cells suggested that one or more of the larger VPg-containing polypeptides initiates RNA synthesis (7, 11). We report here that free VPg as well as a discrete molecule with the structure VPg-pUpU is present in poliovirus-infected cells. MATERIAL AND METHODSPoliovirus was grown in suspension-cultures of HeLa cells. Cells were labeled with 32Pi (500 gCi/ml; 1 Ci = 37 GBq) 1-5 hr after infection or with [3H]leucine (350 ,Ci/ml) 3.5-5.5 hr after infection as described (7). Cell lysates were prepared as described (7) except they were not precleared with antivirion serum, and 8 x 107 cells were lysed with 9 ml of phospholysis buffer (1% Triton X-100/0.5% sodium deoxycholate/0. 1% NaDod-S04/5 mM EDTA/0. 1% aprotinin in phosphate-buffered saline). 32P-Labeled virion RNA was isolated as described (12) from virions collected by centrifugation during preparation of the cell lysates. Synthetic VPg and coupled VPg-bovine serum albumin were gifts from Margaret Baron (7). 125I-Labeled VPg was prepared by iodinating synthetic VPg on tyrosine with Nam25I and Iodo-Beads (Pierce) as recommended by...

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confidence: 99%

mentioning

confidence: 99%

Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.

Crawford¹,

Baltimore²

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…This failure has led us to speculate that VPg is generated from precursor molecule(s) by proteolytic cleavage only at the initiation of RNA synthesis (5). Three putative precursor molecules for VPg (P3-9, 3b/9, and X/9) have been identified by radioimmunoassays (3,4,6) and by amino acid sequence analysis (4,6). All three VPg-containing polypeptides are membrane bound, presumably because they contain a nonpolar sequence of 22 amino acids (4,6).…”

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confidence: 99%

Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus.

Takegami¹,

Kühn²,

Anderson³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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115

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A small nucleotidyl-protein has been synthesized in vitro in a membrane fraction of poliovirus-infected HeLa cells. Analyses of the nucleotides and polypeptide have shown that the nucleotidyl-protein is VPg-pUpU: the genome-linked protein of poliovirion RNA covalently bound to the first two 5'-terminal nucleotides of poliovirus RNA. Synthesis of VPg-pUpU in vitro was sensitive to nonionic detergent. We suggest that VPg-pUpU is part of the initiation complex in poliovirus RNA replication in a membranous environment.

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“…2C). This protein was also found in a free state, not linked to RNA, as has been shown for other viral systems (Takegami et al, 1983). The existence of free VPg within the RCs could be demonstrated by its ability to become uridylylated in the presence of radioactive UTP (Fig.…”

Section: Discussionmentioning

confidence: 64%

“…3A, lane 2). In contrast uridylylation of both VPg and its precursor has been reported for poliovirus (Takegami et al, 1983). Interestingly, exogenous recombinant VPg added to the RCs seems to be accessible to the uridilylation machinery of the RCs membranous preparation (Fig.…”

Section: Discussionmentioning

confidence: 92%

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells

et al. 2008

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a b s t r a c tRabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B-and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

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Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

Cited by 136 publications

References 24 publications

Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.

Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.

Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells

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