New insights into inhibition of human immunodeficiency virus type 1 replication through mutant tRNA Lys3
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RESEARCH
Open Access
New insights into inhibition of human immunodeficiency virus type 1 replication through mutant tRNALys3 Chengxiang Wu1,2, Vivek R Nerurkar3 and Yuanan Lu1*
Abstract Background: Host cellular tRNALys3 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 tRNALys3 with 7nucleotide 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 tRNALys3 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 tRNALys3 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 tRNALys3 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 tRNALys3s 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 tRNALys3. To increase the expression of mutant tRNALys3, 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 tRNALys3 and its target site, and the feasibility of targeting multiple sites within the HIV-1 genome through mutant tRNALys3. Intervention of the HIV-1 genome conversion through mutant tRNALys3 may constitute an effective approach for development of novel therapeutics against HIV-1 replication and HIV-1-associated diseases. Keywords: Mutant tRNALys3, Reverse transcription, HIV-1 inhibition
Background RTion, or the conversion of viral RNA (vRNA) into DNA, is a key step in the life cycle of HIV-1, and it may take place before budding as early as in virus producer cells [1,2]. The reaction is catalyzed by virion-associated reverse transcriptase (RT), and initiated with a cellular primer. Although various primer molecules can be used to initiate RTion in vitro, all retroviruses employ cellular tRNA [3-9]. DNA
* Correspondence: [email protected] 1 Department of Public Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, 1960 East–west Road, Biomed. Bldg, D105, Honolulu, Hawaii 96822, USA Full list of author information is available at the end of the article
sequence analysis of the HIV-1 provirus reveals tRNALys3 to be the primer for HIV-1 RT
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