The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly.
Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys 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 tRNALys.
Javanbakht
Hassan
H
Lady Davis Institute for Medical Research and McGill AIDS Center, Jewish General Hospital, Department of Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada.
Halwani
Rabih
R
Cen
Shan
S
Saadatmand
Jenan
J
Musier-Forsyth
Karin
K
Gottlinger
Heinrich
H
Kleiman
Lawrence
L
D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D017353:Gene Deletion; D015683:Gene Products, gag / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008250:Lysine-tRNA Ligase / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D014162:Transfection
D015683:Gene Products, gag; D016271:Proto-Oncogene Proteins c-myc; D005982:Glutathione Transferase; D008250:Lysine-tRNA Ligase