Reverse transcription assays with transcribed transcripts and vRNA–tRNA complexes isolated from virions

<p><b>Copyright information:</b></p><p>Taken from "The availability of the primer activation signal (PAS) affects the efficiency of HIV-1 reverse transcription initiation"</p><p></p><p>Nucleic Acids Research 2007;35(5):1649-1659.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865047.</p><p>© 2007 The Author(s).</p> () The differences in the anti-PBS and anti-PAS sequences between tRNA and tRNA are boxed in the latter molecule. The anti-PAS and anti-PBS motifs are boxed in gray. The HIV-1 PAS and PBS motifs were mutated to complement the tRNA primer in the mutant PAS/PBS-lys1,2 (M). Long-term culturing of the mutant virus resulted in a revertant virus that gained replication capacity by a point mutation in the PAS motif (R). The U to C mutation stabilizes the interaction of the anti-PAS with the PAS motif. () Reverse transcription initiation of HIV-1 leader RNA transcripts. tRNA primers were annealed to the transcribed wt, mutant (M) and revertant (R) transcripts or the vRNA–tRNA complexes were isolated from the corresponding virions. Primers were extended by addition of HIV-1 RT enzyme and P-dCTP for the extension assay or dTTP, dGTP and P-dCTP for extension. This results in the extension of the tRNA primer with 1 and 5 nt, respectively. The extended primers were separated on a denaturing polyacrylamide gel. The tRNA product runs at a higher position in the gel than the tRNA product due to different base modification