Abstract
Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro. Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure. Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength. Recombinant and virion-derived HIV-1 RTs showed 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 RNA templates were not broken or otherwise unable to support polymerization. Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template. These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausing may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.
Highlights
1 (HIV-1)requires reverse transcriptase (RT) to syn- virus that causes AIDS (Fauci,1988)
Human immunodeficiency virus type 1 (HIV-1) R T was pre-equilibrated with the primer-template, and primer extension reactions were initiated by the addition of dNTPs, MgClZ,and excess of a nonradiolabeled primer-template trapT. hterapp,oly(rA). oligo(dT), binds R T molecules that dissociate from the preformed RT/primer-template complexes during incubation with dNTPs
Pausing occurred by RT dissociation from primertemplates at pause sites.This is deduced from the observation thatthetotal yields of extendedproductsincreased as a function of time in the absence but not in the presence of trap.The simplest interpretation of these results is that increased yields resulted from multiple cycles of R T dissociation from one or more pause sites followed by reassociation to theunextended 20-mer primers which are presentin large excess
Summary
Replication of HIV-1 requiresreverse transcriptase to con- (0.8 nM) resulted in a distribution of products virtually idenvert the plus-stranded RNgAenome into duplex DNA (Coffin,tical with those in Fig. 2 (data not shown). Rupt DNA polymerization by HIV-1 R T (Huber et al, 1989; Examination of the pause patterns exhibited in panels A, Williams et al, 1990).The pHIV-env clone (Fig. 1)contained B , and C shows that pausing was dependent on the template the Rev response element (RRE) and other portions of the sequence and was independent of primer position. AS observed duringRNA-templated polymerization, the pauses on the HIV-1 DNA templateswere sequence-dependent and occurred at specific template locations regardless of primer position. On the env templates, pausing occurred within thefirst few nucleotides of the predicted hairpin structures of the RRE (Leet al., 1988) at position R9 on the RNA and D4 on the DNA.
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