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J. Biol. Chem., Vol. 281, Issue 28, 19449-19456, July 14, 2006

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In Vitro Characterization of the Interaction between HIV-1 Gag and Human Lysyl-tRNA Synthetase^*

Brandie J. Kovaleski¹, Robert Kennedy¹, Minh K. Hong¹, Siddhartha A. Datta, Lawrence Kleiman^¶, Alan Rein, and Karin Musier-Forsyth²

From the Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, the Retrovirus Assembly Section, HIV Drug Resistance Program, NCI, National Institutes of Health, Frederick, Maryland 21702, and the ^¶Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada

Human immunodeficiency virus type 1 (HIV-1) viral assembly is mediated by multiple protein-protein and protein-nucleic acid interactions. Human tRNA^Lys3 is used as the primer for HIV reverse transcription, and HIV Gag and GagPol are required for packaging of the tRNA into virions. Human lysyl-tRNA synthetase (LysRS) is also specifically packaged into HIV, suggesting a role for LysRS in tRNA packaging. Gag alone is sufficient for packaging of LysRS, and these two proteins have been shown to interact in vitro using glutathione S-transferase pull-down assays. In vitro pull-down assays using truncated constructs have also revealed that residues important for homodimerization of Gag and LysRS are critical for the Gag/LysRS interaction. In this work, we report further in vitro characterization of the interaction between HIV Gag and human LysRS using affinity pull-down assays, fluorescence anisotropy measurements and gel chromatography. An equilibrium binding constant of 310 ± 80 nM was measured for the Gag/LysRS interaction. We also show that capsid alone binds to LysRS with a similar affinity as full-length Gag. Point mutations that disrupt the homodimerization of LysRS and Gag in vitro do not affect their interaction. These results suggest that dimerization of each protein per se is not required for the interaction but that residues involved in forming the homodimer interfaces contribute to heterodimer formation. Gel chromatography studies further support the formation of a Gag/LysRS heterodimer.

Received for publication, February 7, 2006 , and in revised form, May 1, 2006.

^* This work was supported by National Institutes of Health Grant AI054145, National Institutes of Health Postdoctoral Grant GM069339 (to R. K.), and National Institutes of Health Predoctoral Training Grants T32-GM08277 (to M. K. H.) and T32-GM08700 (to B. J. K.). This work was also supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. 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.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455. Tel.: 612-624-0286; Fax: 612-626-7541; E-mail: musier{at}chem.umn.edu.

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