| Summary: | <p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus (HIV-1) preferentially selects tRNA<sup>Lys,3 </sup>as the primer for reverse transcription. HIV-1 can be forced to select alternative tRNAs through mutation in the primer-binding site (PBS) and a region upstream of the PBS designated as the A-loop. Alteration of the PBS and A-loop to be complementary to the 3' terminal nucleotides and anticodon of tRNA<sup>His </sup>results in HIV-1 that can stably utilize this tRNA for replication.</p> <p>Results</p> <p>In the current study, we have investigated the effect that mutations within the A-loop have on the stability of HIV-1 with a PBS complementary to tRNA<sup>His</sup>. For these studies, we have altered the A-loop to be complementary to tRNA<sup>Met</sup>, tRNA<sup>Gln</sup>, tRNA<sup>Ile</sup>, tRNA<sup>Thr </sup>and tRNA<sup>Ser</sup>. All substitutions of the A-loops with the PBS complementary to tRNA<sup>His </sup>resulted in a reduction of infectious virus obtained following transfection of proviral genomes in the 293T cells. Virus replication in SupT1 cells was also impaired as a result of the alteration of the A-loop. Viruses with the A-loop complementary to tRNA<sup>Lys,3 </sup>and tRNA<sup>Ser </sup>reverted to utilize tRNA<sup>Lys,3 </sup>following <it>in vitro </it>replication. In contrast, viruses with the A-loop complementary to the other tRNAs remained stable and continued to use tRNA<sup>His</sup>. RNA modeling of the stem-loop structure revealed that nucleotides were displayed on the loop region that could potentially interact with the anticodon of tRNA<sup>His</sup>. To further explore the effects of the A-loop mutations on virus replication, the A-loops complementary to tRNA<sup>Ser </sup>or tRNA<sup>His </sup>were cloned into the wild type genome with the PBS complementary to tRNA<sup>Lys,3</sup>. Transfection of proviral genomes which contained the wild type PBS and A-loops complementary to tRNA<sup>Ser </sup>or tRNA<sup>His </sup>into 293 T cells did not impact on the production of viruses as measured by p24 antigen ELISA. However, viruses with the A-loop complementary to tRNA<sup>His </sup>had greatly reduced infectivity and replicated poorly in SupT1 compared to the wild type or viruses with the A-loop complementary to tRNA<sup>Ser</sup>.</p> <p>Conclusion</p> <p>These studies demonstrate that complementarity of A-loop region with the anticodon of tRNA<sup>His </sup>has a pronounced effect on the capacity of HIV-1 to utilize tRNA<sup>His </sup>as the primer for reverse transcription. Complementarity between A-loop and anticodon of the tRNA then is important for the selection of the tRNA primer used for reverse transcription.</p>
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