The Role of Pr55 gag in the Annealing of tRNA 3 Lys to Human Immunodeficiency Virus Type 1 Genomic RNA

Abstract

ABSTRACT During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA 3 Lys , is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA 3 Lys 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 tRNA 3 Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55 gag or Pr160 gag-pol . In vivo placement of tRNA 3 Lys 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 Pr55 gag or mutant Pr160 gag-pol , and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55 gag inhibit tRNA 3 Lys placement. The NC mutations in Pr55 gag reduce viral infectivity by 95% and are trans -dominant-negative, i.e., they inhibit genomic placement of tRNA 3 Lys even in the presence of wild-type Pr55 gag . This dominant phenotype may indicate that the mutant Pr55 gag is disrupting an ordered Pr55 gag structure responsible for the annealing of tRNA 3 Lys to genomic RNA.