During human immunodeficiency virus type 1 (HIV-1) assembly, tRNALys isoacceptors are selectively incorporated into virions and tRNA 3 Lys is used as the primer for reverse transcription. We show herein that the tRNA Lys -binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. Lys packaging studies but from tRNA 3 Lys placement studies, which indicate that this protein, and not Pr160 gag-pol , plays a major role in annealing tRNA 3 Lys onto the primer binding site in vitro (9) or in vivo (3).In considering the interactions involved between viral proteins and tRNA Lys during packaging, it must be taken into account that tRNAs have been reported to be channeled from one component of the translational machinery to the next and thus may never be free of this synthetic machinery (26). Such components could involve ribosomes, elongation factors, and aminoacyl-tRNA synthetases. Although it has been shown that elongation factor 1-alpha is packaged into HIV-1 via an interaction with Pr55 gag (5), it is not clear how this protein, which binds to all aminoacylated tRNAs, would confer the ability to selectively package tRNA Lys into the virion. Another tRNAbinding protein in the cytoplasm which is more specific for tRNA Lys is lysyl-tRNA synthetase (LysRS). This enzyme is an attractive candidate for interacting specifically with viral proteins and may play a role in the transport of the three tRNA Lys isoacceptors into the virions. In this work, we will show that during viral assembly, LysRS is in fact nonrandomly packaged into HIV-1 through interaction with Pr55 gag and that a truncated LysRS species associated with selective tRNA Lys packaging is found within the virion. MATERIALS AND METHODSPlasmid construction. SVC21.BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA and was a gift from E. Cohen, University of Montreal. pSVGAG-RRE-R and pSVFS5TprotD25G, which code for Gag and unprocessed Gag-Pol, respectively, have been described previously (24,25). Viral production from either of these two plasmids, which contain the Rev response element (RRE), requires cotransfection with a Rev protein expression vector, such as pCMV-REV. Thus, cotransfection of pSVGAG-RRE-R with pCMV-REV is required to produce virus-like particles containing the unprocessed Pr55 gag precursor protein. In this study, pSVSF5TprotD25G was cotransfected with SVC21P31L, a plasmid coding for HIV-1 proteins including Gag and Rev, but not for stable Gag-Pol. The construction of the mutants SVC21 Dr2 and SVC21 P31L has been described previously (12,19).Cell lines. COS7 cells were maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum and antibiotic. H9, PLB, CEMss, and U937 cell lines were grown in RPMI 1640 with 10% fetal bovine serum and antibiotic.Production of wild-type and mutant HIV-1 virus. Transfection of COS7 cells with the above plasmids by the calcium phosphate method was done as previously described (18). Viruses were isolated from COS7 cell culture mediu...

The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known. The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.

In this work, we probe the role of the anticodon in tRNA recognition by human lysyl-tRNA synthetase (hLysRS). Large decreases in aminoacylation efficiency are observed upon mutagenesis of anticodon positions U35 and U36 of human tRNA(Lys,3). A minihelix derived from the acceptor-TPsiC stem-loop domain of human tRNA(Lys,3)was not specifically aminoacylated by the human enzyme. The presence of an anticodon-derived stem-loop failed to stimulate aminoacylation of the minihelix. Thus, covalent continuity between the acceptor stem and anticodon domains appears to be an important requirement for efficient charging by hLysRS. To further examine the mechanism of communication between the critical anticodon recognition elements and the catalytic site, a two piece semi-synthetic tRNA(Lys, 3)construct was used. The wild-type semi-synthetic tRNA contained a break in the phosphodiester backbone in the D loop and was an efficient substrate for hLysRS. In contrast, a truncated variant that lacked nucleotides 8-17 in the D stem-loop displayedseverely reduced catalytic efficiency. The elimination of key tRNA tertiary structural elements has little effect on anticodon-dependent substrate binding but severely impacts formation of the proper transition state for catalysis. Taken together, our studies provide new insights into human tRNA structural requirements for effective transmission of the anticodon recognition signal to the distal acceptor stem domain.