Effect of Lysyl-tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase

In human immunodeficiency virus-1 (HIV-1), reverse transcriptase (RT) is encoded as a 66 kDa protein, p66, in the Gag-Pol polyprotein. This protein is proteolytically cleaved by HIV-1 protease (PR) to finally generate a mature RT that is a heterodimer, composed of a p66 subunit and a p66-derived 51 kDa subunit, p51. In our prior work, we demonstrated that tRNA^Lys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNA^Lys3 is known to be recruited to the virus by forming a complex with lysyl-tRNA synthetase (LysRS). Herein, we tested whether LysRS can have an effect on RT maturation in vitro. Importantly, our data show no significant differences in RT maturation in the presence of LysRS. Furthermore, no apparent p66/66 interaction with LysRS was observed. Although PR cleaved LysRS, it did not immediately release tRNA^Lys3 from LysRS. Thus, we conclude that a free fraction of tRNA^Lys3, which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNA^Lys3 from LysRS. Given that only transient tRNA^Lys3-p66/p66 interaction is needed for efficient RT maturation, a small amount of free tRNA may be sufficient for this process. These studies reveal molecular level insights into RT maturation and will be useful for the design of cellular/viral experiments to better understand the role of tRNA in HIV-1 replication.