Abstract
We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5' transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.
Highlights
Strand transfer is an essential step in the replication of retroviruses, including that of the human immunodeficiency virus type 1 (HIV-1).1 It involves the movement of a primer from one position on the viral genome and reannealing to a different position
We previously found a positive correlation between sequencedependent pausing of reverse transcriptase (RT) and the frequency of strand transfer, using HIV-nef-derived templates [8]
Our original hypothesis for the mechanism of internal strand transfer in the HIV transactivation response region (TAR) region was that the stable secondary structure halts the RT, which extensively degrades the RNA donor around the pause site, freeing the nascent DNA primer for transfer
Summary
Strand transfer is an essential step in the replication of retroviruses, including that of the human immunodeficiency virus type 1 (HIV-1). It involves the movement of a primer from one position on the viral genome and reannealing to a different position. During synthesis by the HIV reverse transcriptase (HIV-RT) in vitro [8] It appears that strand transfer resulting in mutations or homologous recombination is a likely source of some of the genetic variation that produces quasispecies of HIV. This genomic alteration impacts the efficacy of antiviral therapy against HIV, presumably by initiating the emergence of drugresistant viral strains carrying mutations in their genome. Internal strand transfers require the RNase H activity of HIV-RT to degrade the RNA template, called the donor, originally hybridized to the extended primer [9]. It would be of interest to measure strand transfer from a donor template with a large hairpin structure
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