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

Semler, Bert L.; Anderson, Carl W.; Hanecak, Ronnie; Dorner, Lydia F.; Wimmer, Eckard

doi:10.1016/0092-8674(82)90358-0

Cited by 147 publications

(113 citation statements)

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“…DISCUSSION We have detected free cytoplasmic VPg and VPg-pUpU in poliovirus-infected cells. Previous attempts to find free VPg (7,8) probably failed because VPg leached from the polyacrylamide gels during fluorography or fixation. We minimized this problem by omitting the fixation step and by rapid fluorography of gels containing [3H]leucine-labeled material using Enlightening.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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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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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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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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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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“…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).…”

mentioning

confidence: 99%

“…1 and references therein). Studies to support this hypothesis have been complicated due to a failure to detect free VPg in the infected cell by isotope labeling (2) or by radioimmunoassays (3,4). 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).…”

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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.

139

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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Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

1995

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Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

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

Cited by 147 publications

References 20 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.

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

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