New insights into inhibition of human immunodeficiency virus type 1 replication through mutant tRNA(Lys3)

BACKGROUND: Host cellular tRNA(Lys3) is exclusively utilized by human immunodeficiency virus type 1 (HIV-1) as a primer for the replication step of reverse transcription (RTion). Consequently, the priming step of HIV-1 RT constitutes a potential target for anti-HIV-1 intervention. Previous studies indicated that a mutant tRNA(Lys3) with 7-nucleotide substitutions in the 3′ terminus resulted in aberrant HIV-1 RTion from the trans-activation response region (TAR) and inhibition of HIV-1 replication. However, the mutant tRNA(Lys3) also directed HIV-1 RTion from the normal primer-binding site (PBS) with potentially weakened anti-HIV-1 activity. To achieve improved targeting of HIV-1 RTion at sites not including the PBS, a series of mutant tRNA(Lys3) with extended lengths of mutations containing up to 18 bases complementary to their targeting sites were constructed and characterized. RESULTS: A positive correlation between the length of mutation in the 3′ PBS-binding region of tRNA(Lys3) and the specificity of HIV-1 RTion initiation from the targeting site was demonstrated, as indicated by the potency of HIV-1 inhibition and results of priming assays. Moreover, two mutant tRNA(Lys3)s that targeted the IN-encoding region and Env gene, respectively, both showed a high anti-HIV-1 activity, suggesting that not only the TAR, but also distant sites downstream of the PBS could be effectively targeted by mutant tRNA(Lys3). To increase the expression of mutant tRNA(Lys3), multiple-copy expression cassettes were introduced into target cells with increased anti-HIV-1 potency. CONCLUSIONS: These results highlight the importance of the length of complementarity between the 3′ terminus of the mutant tRNA(Lys3) and its target site, and the feasibility of targeting multiple sites within the HIV-1 genome through mutant tRNA(Lys3). Intervention of the HIV-1 genome conversion through mutant tRNA(Lys3) may constitute an effective approach for development of novel therapeutics against HIV-1 replication and HIV-1-associated diseases.