MECHANISTIC STUDIES OF EARLY PAUSING EVENTS DURING INITIATION OF HIV-1 REVERSE TRANSCRIPTION

We have investigated the role of sequences that surround the primer bi nding site (PBS) in the reverse transcriptase-mediated initiation of ( -) strand DNA synthesis in human immunodeficiency virus type I. In com parisons of reverse transcription initiated from either the cognate pr imer tRNALys.3 or a DNA primer D-Lys.3, bound to PBS sequences, we obs erved that a +3 pausing site occurred in both circumstances, However, the initiation reaction with tRNALys.3 was also characterized by a pau sing event after incorporation of the first nucleotide, Alteration of sequences at the 5'-end instead of the 3'-end of the PBS resulted in e limination of the +3 pausing site, suggesting that this site was templ ate sequence-dependent. In contrast, the pausing event at the +1 nucle otide position was still present in experiments that employed either o f these mutated RNA templates. The mutations at the 5'-end of the PBS also caused a severely diminished rate of initiation and the strong ar rest of reactions at the fl stage when tRNALys.3 was used as primer. T herefore, we propose that the ct pausing event is an initiation-specif ic event in regard to reactions primed by tRNALys.3 and that sequences at the 5'-end of the PBS may facilitate the release of reverse transc ription from initiation to elongation.