The Human immunodeficiency virus type 1 (HIV-1) is a retrovirus with a genome composed of two molecules of single stranded RNA. The reverse transcriptase encoded by HIV-1 uses the cellular tRNA3Lys to prime the replication of its RNA genome into a proviral DNA. The tRNA3Lys is packaged into the viral particles during their assembly; the cellular lysyl-tRNA synthetase (LysRS) is involved in this mechanism as a co-carrier of tRNA3Lys.In human, there are two forms of LysRS, a cytoplasmic form (cLysRS) and a mitochondrial form (pmLysRS) that will be maturated into mLysRS after translocation into the mitochondrion. Both LysRS arise from the same gene by alternative splicing. It was demonstrated that only the mitochondrial species is present in the viral particles.We established a model of the protein-protein interactions which are implied in the formation of the packaging complex of tRNA3Lys. By searching for interactions of the viral precursors Gag and GagPol with the LysRS species and their domains, we demonstrated that only the Pol domain of the GagPol precursor has the capacity to interact with LysRS. The transframe (TF) and integrase (IN) domains of the Pol region of the polyprotein GagPol are required for association of LysRS with GagPol. This association is mediated by the catalytic domain of the enzyme. The selectivity of the packaging of the mitochondrial species of LysRS but not of its cytoplasmic species would rest on the cellular compartmentalization of these two enzyme forms. To establish at which step the mitochondrial LysRS is packaged, either as the pmLysRS precursor before its mitochondrial translocation, or after as the mature mLysRS, we determined the site of maturation of the pmLysRS precursor, then we characterized both mitochondrial forms of LysRS, by determining their kinetic parameters and their affinity for tRNA3Lys. Whereas the pmLysRS species did not form a stable complex with tRNA, the mature pmLysRS species did. Thus, mLysRS is the only LysRS species which could be implied in the transport of tRNA3Lys into the viral particles during the budding step. In vitro, the interaction GagPol:LysRS is not specific for the mLysRS species, but only the mitochondrial LysRS is packaged into the viral particles. We determined if another viral protein could impact the specificity of mLysRS packaging. We showed that the auxiliary proteins Rev and Vpr have the capacity to interact with LysRS but this intercation is not recovered in the context of the GagPol:mLysRS:tRNA3Lys packaging complex. These data suggest that the different forms of LysRS might regulate the activity of Vpr and Rev at other steps of the viral cycle.
