Reverse transcription in HIV-1 (human immunodeficiency virus type 1) is initiated from a tRNA, tRNALys3, that is annealed to the primer binding site (PBS) in the 5' region of viral RNA. This tRNA, along with the other major tRNALys isoacceptors, tRNALys1,2 , is selectively packaged into HIV-1 during its assembly. The formation of a tRNALys packaging/annealing complex is believed to involve the interaction between a Gag/GagPol/viral complex with a lysyl-tRNA synthetase (LysRS)/tRNALys complex, with Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In fact, Gag particles alone will package LysRS, but GagPol, which binds tRNA Lys, is also required for incorporation of the tRNALys. / The model we propose for the tRNALys packaging/annealing complex predicts a possible interaction between LysRS and Pol sequences in GagPol, which might facilitate transfer of tRNALys3 from LysRS to the reverse transcriptase (RT) thumb domain where tRNALys3 binds. In this work, we demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. Since these RT domains are not required for tRNALys packaging into HIV-1, the LysRS/Pol interaction is probably not involved in the transfer of tRNALys3 to RT. The LysRS/Pol interaction may instead be involved in tRNALys3 annealing since the connection domain in RT has been found to be required for this process. Also, since an interaction has been reported between Gag and Pol sequences in GagPol, we also investigated whether the Gag/LysRS/Pol interaction played an important role in stabilizing the Gag/Pol interaction, and found, using siRNA to LysRS, that it did not. / tRNALys3 annealing to viral RNA is promoted by nucleocapsid sequences in Gag and by mature NCp7, and we have examined the roles of Gag and NCp7 in this process. Gag- and NC-facilitated tRNALys3 annealing to HIV-1 RNA were measured both in vivo and in vitro, indirectly by the ability of annealed tRNALys3 to prime reverse transcription, and directly by measuring the occupancy of the PBS by tRNALys3. While tRNALys3 annealing can be carried out by both Gag and NCp7, exposure (in vivo or in vitro) of the tRNALys3/viral RNA complex to NCp7 is required for optimum placement of the tRNALys3. This is indicated by 1) tRNALys3's reduced ability to incorporate the first dNTP, dCTP, and 2) its more ready displacement from the PBS by DNA synthesized from a downstream primer. / It has been previously demonstrated that APOBEC3G (A3G) can inhibit tRNA Lys3 annealing to viral RNA, and we have used A3G to further dissect the roles of Gag and NCp7 in annealing, both in vitro and in vivo. Experiments studying how APOBEC3G (A3G) inhibits tRNA Lys3 annealing indicate that in protease-positive viruses, Gag-facilitated tRNALys3 annealing may only playa minor role. In vivo and in vitro, A3G only inhibits NCp7-facilitated annealing, and not Gag-facilitated annealing. Nevertheless, while Gag is able to show 70-80% of the annealing efficiency of NCp7 in a protease-negative virus, A3G can reduce annealing efficiency in protease-positive viruses to 40%. This appears to be due to the fact that, in vitro, the presence of NCp7 makes prior Gag-facilitated annealing susceptible to A3G. This suggests that in wild type viruses, any Gag-facilitated annealing of tRNALys3 to viral RNA that does occur is altered through an A3G-susceptible re-annealing by NCp7.

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