Mutation of the methylated tRNA(3)(Lys) residue A58 disrupts reverse transcription and inhibits replication of human immunodeficiency virus type 1

Cellular tRNA(3)(Lys) serves as the primer for reverse transcription of hum an immunodeficiency virus type 1 (HIV-1). tRNA(3)(Lys) interacts directly w ith HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to i nitiate reverse transcription. Residue A58 of tRNA(3)(Lys), which lies outs ide the PBS-complementary region, is posttranscriptionally methylated to fo rm 1-methyladenosine 58 (M(1)A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse tra nscription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we inve stigated the role of tRNA(3)(Lys) residue A58 in the replication cycle of H IV-1 in living cells. We have developed a mutant tRNA(3)(Lys) derivative, t RNA(3)(Lys)A58U, in which A58 was replaced by U. This mutant tRNA was expre ssed in CEM cells. We demonstrate that the presence of M1A58 is necessary f or the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M1A58 allows RT to read the tRNA sequences beyond resi due 58. In addition, we show that replacement of M1A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRN A primer residue A58 in the reverse transcription process. Inhibition of re verse transcription with mutant tRNA primers constitutes a novel approach f or therapeutic intervention against HIV-1.