Abstract
BackgroundHost 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 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 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.ResultsA 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.ConclusionsThese 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.
Highlights
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)
In designing the mutant tRNALys3, we extended the length of mutations in the 3′ terminal primer-binding site (PBS)-binding region to enhance their binding specificity and efficiency of directing the RTion of HIV-1 to new targeting sites
RTion priming tests indicated that these mutants were as effective as others targeting the trans-activation response region (TAR) (Figure 7B) with high specificity and efficiency, showing no detectable priming activity from the PBS. Both Median tissue culture infective dose (TCID50) assay and HIV-1 challenging tests indicated these mutants could lead to inhibition of HIV-1 infection to similar potencies as Mt13TD (Figure 3A and 3B). These findings clearly suggest that the mutant tRNALys3-mediated inhibition of HIV-1 replication is not limited to targeting sites upstream the PBS and other portions of the viral RNA (vRNA) could be effectively targeted
Summary
Host cellular tRNALys is exclusively utilized by human immunodeficiency virus type 1 (HIV-1) as a primer for the replication step of reverse transcription (RTion). An 18-nucleotide residue at the 3′ terminus of the tRNALys anneals complementarily to the PBS of vRNA, and primes template-dependent DNA synthesis [12]. The (−)ssDNA is released and anneals to the 3′ terminus of the vRNA, and primes further (−)strand DNA synthesis and generates a full-length (−)strand DNA that is used as a template for (+)strand DNA synthesis. Along with (−)strand DNA synthesis, RNaseH degrades the RNA template with the exception of two polypurine tracts (PPTs) that resist cleavage: one immediately upstream of the U3 region (3′-PPT) and the other at the center of the vRNA (cPPT). These PPTs are responsible for priming (+)strand DNA synthesis. For alpha and gamma-retroviruses and lentiviruses, these obligatory steps in genome conversion are chaperoned by a major virion protein of the inner core, the nucleocapsid protein encoded by Gag that serves as a key cofactor of the RT enzyme [15,16,17,18,19,20,21]
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