In vitro studies on tRNA annealing and reverse transcription with mutant HIV-1 RNA templates

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semist able stem-loop structure, the US-PBS hairpin, which occludes part of the tR NA primer binding site (PBS). In previous studies, we demonstrated that mut ations that alter the stability of the U5-PBS hairpin inhibit virus replica tion. A reverse transcription defect was measured in assays with the virion -extracted RNA-tRNA complexes. We now extend these studies with in vitro sy nthesized wild-type and mutant RNA templates that were tested in primer ann ealing and reverse transcription assays. The effect of annealing temperatur e and the presence of the viral nucleocapsid protein on reverse transcripti on was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to sta ble template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA prim ed reactions. The inhibitory effect of template structure on tRNA-primed re verse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex d iffers from the virion-extracted viral RNA-tRNA complex.