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J Mol Biol. 2009 Dec 11;394(4):694-707. doi: 10.1016/j.jmb.2009.10.036. Epub 2009 Oct 21.

Factors that determine the efficiency of HIV-1 strand transfer initiated at a specific site.

Author information

1
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

Abstract

Human immunodeficiency virus-1 employs strand transfer for recombination between two viral genomes. We have previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long-distance propagation of the hybrid. Evaluation was performed on a unique substrate, with a short gap serving as a precreated invasion site. In our current work, this substrate provided an opportunity for us to test what factors influence a specific invasion site to support transfer, and to distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or polymerization-independent mode. Polymerization-dependent and polymerization-independent RNase H were found to be important in creating efficiently used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a precreated invasion site in the presence of NC, were stimulated by polymerization-dependent, but not polymerization-independent, RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer even if synthetic progress of reverse transcriptase was intentionally slowed, consistent with strand exchange by random walk in which rate declines precipitously with distance.

PMID:
19853618
PMCID:
PMC2860860
DOI:
10.1016/j.jmb.2009.10.036
[Indexed for MEDLINE]
Free PMC Article

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