The Role of Pr55gag in the Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Genomic RNA

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American Society for Microbiology

RESUMO

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55gag or Pr160gag-pol. In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55gag or mutant Pr160gag-pol, and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55gag inhibit tRNA3Lys placement. The NC mutations in Pr55gag reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55gag. This dominant phenotype may indicate that the mutant Pr55gag is disrupting an ordered Pr55gag structure responsible for the annealing of tRNA3Lys to genomic RNA.

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