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Journal of Virology, May 1999, p. 4485-4488, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Shan Cen,1 Yue Huang,1,2 Ahmad Khorchid,1,3 Jean-Luc Darlix,4 Mark A. Wainberg,1,2,3 and Lawrence Kleiman1,2,3,*

Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital,1 and Departments of Medicine3 and Immunology and Microbiology,2 McGill University, Montreal, Quebec, Canada H3T 1E2, and LaboRetro, Unite de Virologie Humaine, INSERM U412, Ecole Normale Superieure de Lyon, 69364 Lyon Cedex, France4

Received 26 October 1998/Accepted 9 February 1999

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.

* Corresponding author. Mailing address: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-7502. E-mail: md26{at}musica.mcgill.ca.

Journal of Virology, May 1999, p. 4485-4488, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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