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

Abstract

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...

Biologically Active Transcripts of a Large Satellite RNA from Arabis Mosaic Nepovirus and the Importance of 5' End Sequences for its Replication

“…Interestingly, both recombinant coxsackievirus B3 (CVB3) cDNA and transcripts of CVB3 lacking the two Y-most uridine residues present in authentic virion RNA, are infectious (Klump et al, 1990), and during replication in transfected cells the authentic Y-terminal sequence is produced. The finding of Y-terminal self-correction with CVB is consistent with the suggestion that VPg-pUpU is a component of the initiation complex for poliovirus RNA (Crawford & Baltimore, 1983;Takegami et al, 1983;Takeda et al, 1986;Wimmer et al, 1987). Our experience with satellite RNA, though not identical, is perhaps analogous and may imply that VPg plus a few nucleotides at the 5' terminus (which possibly arise as a result of the replication of the ArMV genomic RNAs) serve as a primer for plus-strand synthesis, independent of the complementary terminal residues on the minusstrand template.…”

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

“…Analysis of the Uridylylated VPg Products-Free VPg, carrying more than one covalently linked UMP (VPg pUpU), has been found in PV-infected cells (30). This precursor to elongation is most likely synthesized independently of the elongation reaction.…”

A “Slide-back” Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus

“…Therefore the termination of elongation by PV 3D pol is likely due to some structural characteristics of the polymerase that are a prerequisite of the nucleotidylylation reaction. Such abortive synthesis of VPgpUpU also occurs in vitro on a poly(A) template (9), in crude replication complexes isolated from poliovirus-infected cells (41,42), and in poliovirus-infected HeLa cells in vivo (43). It is interesting to note that picornaviral RNA polymerases are mechanistically similar to DNA-dependent RNA polymerases in that during the initiation phase RNA synthesis is aborted.…”