Abstract
According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.
Highlights
HIV1 is a retrovirus and the causative agent of AIDS
Strand Displacement Slows human immunodeficiency virus reverse transcriptase (HIV-reverse transcriptase (RT))-directed Synthesis— HIV-RT was allowed to carry out DNA synthesis on the substrates used to study strand displacement (Fig. 1B) in a time course reaction (Fig. 2)
All the enzymes on substrate B dissociated before adding 100 nucleotides. These results show a decrease in processivity, the number of nucleotides added per binding of RT to the polynucleotide substrate, when the enzyme performs strand displacement synthesis
Summary
HIV1 is a retrovirus and the causative agent of AIDS. Retroviruses convert their RNA genomes into proviral DNA by the process of reverse transcription (reviewed in Whitcomb and Hughes (1992)). Transfer in this case is thought to be facilitated by the removal of the tRNA primer which allows the complementary sequences spanning the PBS site, at the ends of the (ϩ)- and (Ϫ)-strands, to circularize and complete their synthesis (Fig. 1A) After this circularization step in HIV, polymerization displaces about 637 nucleotides (Ratner et al, 1985) to generate the long terminal repeats containing the duplications of the U3, R, and U5 regions. Strand displacement was proposed to be involved in a new model for the second template switch in reverse transcription (Li et al, 1993) In this model, synthesis from the central PPT displaces the plus strong stop DNA. We examine the ability of HIV-RT to displace a 634-nucleotide-long segment of DNA containing the natural LTR sequences in vitro
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