Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo.
In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.
Stewart
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Department of Microbiology, University of Washington, Seattle 98195.
Vogt
V M
VM
D000595:Amino Acid Sequence; D001354:Avian Leukosis Virus / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D015685:Fusion Proteins, gag-pol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism; D014771:Virion / Q000737:chemistry
D015685:Fusion Proteins, gag-pol; D011994:Recombinant Proteins; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase; D010450:Endopeptidases