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.
Highlights
Template switching during reverse transcription promotes recombination in retroviruses
To distinguish the roles of the proteins from those of the substrate, we examined each step in the transfer mechanism in the presence and absence of NC, using HIV-1 and equine infectious anemia virus (EIAV) reverse transcriptase (RT)
HIV-1 RT and EIAV RT Show Similar Pattern of Pausing— Donor RNA DI contains a strong hairpin, and when used in conjunction with acceptor AI-2 most transfers are completed before the last nucleotide on the donor is copied [54]
Summary
Template switching during reverse transcription promotes recombination in retroviruses. 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. Because RT has DNA polymerase and RNase H activities within the same molecule [39, 40], it was suggested that pausing would temporarily inhibit nucleotide addition, but it would not affect the nuclease function This would result in degradation of the RNA donor template in the region of pausing and subsequent exposure of the cDNA. Base pairing between a homologous acceptor molecule and the cDNA would result in a transfer event (36 – 38) and polymerization would resume on the acceptor template In support of this model, RNase H activity has been found to be necessary for strand transfers.
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