Panoply™ Human KARS Knockdown Stable Cell Line

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein, which is subsequently proteolytically processed by HIV-1 protease (PR) to form a mature heterodimer composed of p66 and its derivative 51 kDa subunit, p51. Previous studies have shown that the binding of tRNA^Lys3 to p66/p66 leads to a conformational change in the ribonuclease H (RNH) domain of RT, thereby promoting the efficient cleavage of p66 into p51 by PR. Here, researchers used nuclear magnetic resonance (NMR) spectroscopy to characterize the tRNA^Lys3-induced conformational changes in the p66/p66 RNH domain. Furthermore, the importance of tRNA^Lys3 in RT maturation was confirmed at the cellular level by modulating the levels of lysyl-tRNA synthetase, an enzyme that affects the binding of tRNA^Lys3 to the virus. The researchers also used non-nucleoside RT inhibitors to modulate the p66 dimer-monomer equilibrium and monitored the resulting structural changes. Collectively, these data provide unique insights into how conformational changes in p66/p66 drive PR cleavage.

To investigate the role of tRNA^Lys3 in the maturation process of reverse transcriptase (RT), researchers constructed KARS knockdown 293T cells and then transfected these cells with a full-length infectious HIV-1 molecular clone (HIV-1_LAI) (Figure 1A). They observed intracellular RT accumulation, a phenomenon not seen in control cells (Figures 8B and 8C). Interestingly, p66 protein accumulated in KARS knockdown cells, but HIV-1 protease (PR) did not appear to efficiently process it into p51 (Figures 1B and 1C), suggesting that tRNA^Lys3 may affect RT maturation in the cellular environment. Compared to control cells, KARS knockdown resulted in a significant reduction in viral particle production (as measured by p24 quantification) (Figure 1D). However, the p66:p51 ratio in the viral particles produced by KARS knockdown cells was 1:1 (Figures 1E and 1F), and there was no difference in the relative infectivity of the viruses produced by the control and KARS knockdown cell lines (measured in TZM-bL cells) (Figure 1G). These observations may indicate that tRNA^Lys3 plays a role in viral assembly. Due to the low viral yield from these cells, the researchers were unable to measure tRNA^Lys3 levels in the viral particles produced by the KARS knockdown cell line.

Figure 1. siRNA-Mediated Knockdown of KARS in 293T Cells. (Slack R L, et al., 2019)