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J. Biol. Chem., Vol. 278, Issue 30, 27644-27651, July 25, 2003

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The Interaction between HIV-1 Gag and Human Lysyl-tRNA Synthetase during Viral Assembly^*

Hassan Javanbakht  , Rabih Halwani  , Shan Cen , Jenan Saadatmand  , Karin Musier-Forsyth ¶, Heinrich Gottlinger || and Lawrence Kleiman   ** 

From the Lady Davis Institute for Medical Research and McGill AIDS Center, Jewish General Hospital, Departments of Medicine and ^**Microbiology & Immunology, McGill University, Montreal, Quebec H3T 1E2, Canada, ^¶Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and ^||Department of Cancer, Immunology and AIDS, Dana Farber Cancer Institute, Boston, Massachusetts 02115

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNA^Lys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag·LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208–259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNA^Lys.

Received for publication, February 20, 2003 , and in revised form, April 28, 2003.

^* This work was supported in part by grants from the Canadian Institutes for Health Research and the Canadian Foundation for AIDS Research. This work was performed by H. Javanbakht in partial fulfillment of the Ph.D. degree at McGill University, Montreal, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Lady Davis Institute for Medical Research-Jewish General Hospital, 3755 Cote St. Catherine Rd., Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260; Fax: 514-340-7502; E-mail: lawrence.kleiman{at}mcgill.ca.

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