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.