Role of the Reverse Transcriptase, Nucleocapsid Protein, and Template Structure in the Two-step Transfer Mechanism in Retroviral Recombination

Abstract

Template switching during reverse transcription promotes recombination in retroviruses. Efficient switches have been measured in vitro on hairpin-containing RNA templates by a two-step mechanism. Pausing of the reverse transcriptase (RT) at the hairpin base allowed enhanced cleavage of the initial donor RNA template, exposing regions of the cDNA and allowing the acceptor to base pair with the cDNA. This defines the first or docking step. The primer continued synthesis on the donor, transferring or locking in a second step. Here we determine the enzyme-dependent factors that influence template switching by comparing the RTs from human immunodeficiency virus, type 1 (HIV-1), and equine infectious anemia virus (EIAV). HIV-1 RT promoted transfers with higher efficiency than EIAV RT. We found that both RTs paused strongly at the base of the hairpin. While stalled, HIV-1 RT made closely spaced cuts, whereas EIAV RT made only a single cut. Docking occurred efficiently at the multiply cut but not at the singly cut site. HIV-1 nucleocapsid (NC) protein stimulated strand transfers. It improved RNase H activity of both RTs. It allowed the EIAV RT to make a distribution of cuts, greatly stimulating docking at the base of the hairpin. Most likely, it also promoted strand exchange, allowing transfers to be initiated from sites throughout the hairpin. Minor pause sites beyond the base of the hairpin correlated with the locking sites. The strand exchange properties of NC likely promote this step. We present a model that explains the roles of RNase H specificity, template structure, and properties of NC in the two-step transfer reaction.

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

“…Together, these control reactions indicated that the greater RNase H cleavage observed upon addition of NC could be attributed almost entirely to improved annealing of substrates, rather than enhancement of RT RNase H activity, per se. Other studies from our laboratory have demonstrated similar enhancements of template cleavage with the addition of NC (27,31), however, without the extension and cleavage controls employed here it was difficult to distinguish between the two possible scenarios that could explain these effects. Given the data presented here, we suggest that much of the improved RNase H cleavage observed with NC in vitro is the result of improved annealing rather than a direct enhancement of RT RNase H activity by NC.…”

“…Materials-RT was purified as described (27,56) except that p66 and p51 subunits were purified separately and mixed in equal amounts prior to dialysis. DNA oligonucleotides and RNA templates T2 and T3 were obtained from Integrated DNA Technologies (Coralville, IA).…”

“…NC has also been suggested to influence the RNase H activity of RT. In several studies, NC was observed to increase overall RNase H cleavage during primer extension (26,27) and suppress the RNase H cleavage defect of an RT mutant (28). Moreover, NC has been shown to enhance secondary cleavages on blunt end substrates (29 -31).…”

“…A series of studies from our group have defined elements of the mechanism of HIV-1 RT-mediated strand transfer in a template system containing a major hairpin sequence derived from the primer-binding site of the equine infectious anemia virus (EIAV) (27,42). In this system, pausing at the base of the hairpin on the first, or donor, template enhances RNase H cleavage by the RT.…”

HIV-1 Nucleocapsid Protein Increases Strand Transfer Recombination by Promoting Dimeric G-quartet Formation

“…However, only 17.12% of the transfer events were located in the gag region in the absence of NC. It should also be noted that NC was not observed to substantially redistribute the general location of transfers with many substrates or alter the general mechanisms of strand transfer (51,52). In addition, we have reported previously a 3.8-fold decrease in the number of the transfer events in the gag hotspot site when the G-runs involved in G-quartet formation were disrupted.…”