No abstract

The primer for reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome is tRNA 3Lys . During assembly of HIV-1 particles, tRNA 3 Lys is taken up from the host cell along with lysyl-tRNA synthetase (LysRS), the tRNA binding protein that specifically aminoacylates the different tRNA Lys isoacceptors. In humans, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means of alternative splicing. Here, we show that polyclonal antibodies directed to the full-length cytoplasmic enzyme equally recognized the two enzyme species. We raised antibodies against synthetic peptides that allowed discrimination between the two enzymes and found that mitochondrial LysRS is the only cellular source of LysRS detected in the virions. These results open new routes for understanding the molecular mechanisms involved in the specific packaging of tRNA 3Lys into viral particles.

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, refered to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNAbinding site. Their mutation into alanine led to a reduced affinity for tRNA 3 Lys or minimalized tRNA mimicking the acceptor-T⌿C stem-loop of tRNA 3Lys and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA Lys networking during translation or to convey tRNA 3 Lys into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNAinteracting factor.