Cooperative methylation of human tRNA(3)(Lys) at positions A58 and U54 drives the early and late steps of HIV-1 replication

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N(1)-methyladenosine modification at tRNA position 58 (m(1)A58); however, the role of m(1)A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m(1)A in HIV-1 infection. We confirmed that tRNA(3)(Lys) m(1)A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m(1)A58-deficient tRNA(3)(Lys) instead of m(1)A58-modified tRNA(3)(Lys). In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2′-O-dimethyluridine at U54 of tRNA(3)(Lys) as a second RT-stop site was supported by conservation of retroviral genome-tRNA(Lys) sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m(1)A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.