TEMPLATE-DIRECTED PAUSING OF DNA-SYNTHESIS BY HIV-1 REVERSE-TRANSCRIPTASE DURING POLYMERIZATION OF HIV-1 SEQUENCES INVITRO

Replication of human immunodericiency virus type 1 (HIV-1) requires re verse transcriptase (RT) to synthesize double-stranded proviral DNA (9 .7 kilobases) through a complex mechanism utilizing both RNA and DNA t emplates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA te mplates derived from the HIV-1 genome using a primer extension assay i n vitro. Analysis of polymerization products on sequencing gels reveal ed strong pauses in synthesis, on both RNA and DNA templates, in homop olymeric nucleotide runs, and at regions of predicted secondary struct ure. Polymerization pauses occurred in runs of template rGs (greater-t han-or-equal-to 4 bases) and rCs (greater-than-or-equal-to 3 bases) du ring minus-strand synthesis on RNA templates, and in most runs (greate r-than-or-equal-to 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates w ithin the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on t emplate sequence and were unaffected by primer positioning, RT concent ration, and ionic strength. Recombinant and virion-derived HIV-1 RTs s howed similar pausing patterns. DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the R NA templates were not broken or otherwise unable to support polymeriza tion. Polymerizations conducted in the presence of a poly(rA).oligo(dT ) trap showed that pausing results from two mechanisms: 1) RT remainin g bound to the primer-template and polymerizing at a greatly reduced r ate, or 2) RT dissociating from the primer-template. These results dem onstrate that specific HIV-1 RNA and DNA template sequences are capabl e of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausi ng may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are re quired to complete proviral DNA synthesis in vivo and that efficient H IV-1 DNA synthesis may require multiple origins.