Directs Specific Initiation of Minus-Strand DNA Synthesis Primed by Human tRNA3 Lys In Vitro: Studies of Viral RNA Molecules Mutated in Regions That Flank the Primer Binding Site

Retroviral reverse transcription starts near the 5 * end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA3 Lys to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of various 5 * viral RNA templates, natural primer tRNA3 Lys or synthetic primer, human immunodeficiency virus type 1 (HIV-1) nu-cleocapsid protein (NCp7), and HIV-1 RT. In the presence of tRNA3 Lys, NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(2)ssDNA], consistent with previous reports. However, specific DNA synthesis was observed only at a NCp7/RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited the synthesis of nonspecific reverse-transcribed DNA products, which are initiated because of self-priming by RNA templates. In contrast to results obtained with tRNA3 Lys as primer, NCp7 inhibited the synthesis of (2)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rPR)