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

Cellular tRNA(Lys)(3) serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA(Lys)(3) interacts directly with 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 initiate reverse transcription. Residue A58 of tRNA(Lys)(3), which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 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 transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA(Lys)(3) residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA(Lys)(3) derivative, tRNA(Lys)(3)A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M(1)A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M(1)A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M(1)A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.

Defective lentiviral vectors are efficiently trafficked by HIV-1 and inhibit its replication.

Gene therapy against HIV infection should involve vector-mediated delivery of anti-HIV therapeutic genes into T-lymphocytes and macrophages or, alternatively, hematopoietic progenitors. Transduction of mature cells with defective vectors would have limited success because the vector would disappear with cell turnover. However, if a vector could be trafficked by wild-type HIV, initial transduction of a majority of the population would not be required, as the vector would be able to spread. We describe HIV-1-based lentiviral vectors that are efficiently packaged and trafficked by HIV-1, allowing a small number of cells initially transduced to spread the vector within a nontransduced cell population. We examined whether the presence or absence of the rev gene and the Rev-responsive element (RRE) would have a noticeable effect on the ability of lentiviral vectors to be trafficked and to inhibit HIV-1 replication. We found that replacement of rev/RRE with a constitutive transport element from Mason-Pfizer monkey virus had no apparent effect on trafficking and did not change the intrinsic inhibitory abilities of the vectors. We also constructed a rev/RRE-independent HIV-1-derived vector carrying a trans-dominant negative mutant of HIV-1 Rev, RevM10. This vector was less efficiently trafficked by HIV-1 and, despite the presence of an anti-HIV-1 gene, RevM10, was less efficient at inhibiting HIV-1 replication when introduced into a target T-cell population.