Properties of avian retrovirus particles defective in viral protease.

AUTOR(ES)

Stewart, L

RESUMO

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We constructed an active-site mutation, D37I, in the PR of avian leukosis virus. The D37I mutation was introduced into an infectious DNA clone, and quail cell lines expressing the mutant virus were established. These cell lines produce normal amounts of virus particles, the major internal protein components of which are the uncleaved gag and gag-pol precursors. As in other retroviral systems, the protease-defective virions are noninfectious and retain the "immature" type A morphology as determined by thin-section transmission electron microscopy. The virion cores are stable at nonionic detergent concentrations that completely disrupt wild-type cores. Digestion of mutant virions with exogenous PR in the presence of detergent leads to complete and correct cleavage of the gag precursor but incomplete cleavage of the gag-pol precursor. The protease-defective virions encapsidate normal amounts of genomic RNA and tRNA(Trp) that is properly annealed to the primer-binding site, but some of the genomic RNA remains monomeric. Results from UV cross-linking experiments show that the gag polyprotein of mutant virions interacts with viral RNA and that this interaction occurs through the nucleocapsid (NC) domain. However, within mutant virions the interaction of the NC domain with RNA differs from that of mature NC with RNA in wild-type virions. Reverse transcriptase (RT) activity associated with mutant virions is diminished but still detectable. Digestion of the virions with PR leads to a fivefold increase in activity, but this PR-mediated activation of RT is incomplete. Since in vitro cleavage of the gag-pol precursor is also incomplete, we hypothesize that amino acid sequences N terminal to the reverse transcriptase domain inhibit RT activity.

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