Interactions of reverse transcriptase sequences in Pol with Gag and LysRS in the HIV-1 tRNA Lys3 packaging/annealing complex

Abstract

During HIV-1 assembly, tRNA Lys3, the primer for reverse transcriptase (RT) in HIV-1, is selectively packaged into the virus due to a specific interaction between Gag and lysyl-tRNA synthetase (LysRS). However, while Gag alone will incorporate LysRS, tRNA Lys3 packaging also requires the presence of RT thumb domain sequences in GagPol. The formation of a tRNA Lys3 packaging/annealing complex involves an interaction between Gag/GagPol/viral RNA and LysRS/tRNA Lys, and herein, we have investigated whether the transfer of tRNA Lys3 from LysRS to RT sequences in Pol by a currently unknown mechanism is facilitated by an interaction between LysRS and Pol. We demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. However, cytoplasmic Gag/Pol interactions, detected by either coimmunoprecipitation or incorporation of Pol into Gag viral-like particles, were found to be insensitive to the overexpression or underexpression of LysRS, indicating that a Gag/LysRS/RT interaction is not essential for Gag/Pol interactions. Based on this and previous work, including the observation that the RT connection domain is not required for tRNA Lys3 packaging, but is required for tRNA Lys3 annealing, a model is proposed for a tRNA Lys3 packaging/annealing complex in which the interaction of Gag with Pol sequences during early viral assembly facilitates the retention in budding viruses of both tRNA Lys3 and early Pol processing intermediates, with tRNA Lys3 annealing to viral RNA further facilitated by the LysRS/RT interaction.