Mechanistic analysis of pause site-dependent and -independent recombinogenic strand transfer from structurally diverse regions of the HIV genome.
Retroviral recombinants are generated by strand transfers occurring within internal regions of the viral genome and are a major source of genetic variability. Strand transfer has been linked to "pausing" occurring at secondary structures during synthesis by reverse transcriptase. Yet, weakly structured templates lacking strong pause sites also undergo efficient transfer. In this report, transfer crossover sites on high and low structured templates from the gag-pol frameshift region (GagPol) and the env (Env) regions, respectively, were determined by using a reconstituted in vitro strand transfer assay. The assay tested transfers occurring between a donor and acceptor template over a 150-nucleotide homologous region. The majority of crossovers were in a small 23-nucleotide region near a major pause site on GagPol, clearly indicating a pause-driven mechanism. In contrast, on Env, transfers were more dispersed clustering toward the end of the homologous region. Slowing down polymerization on Env by decreasing the dNTP concentration resulted in crossovers shifting toward the beginning of the homologous region. Removal of a small 38-nucleotide region at the 3'-end of the Env acceptor had a large effect on the level of strand transfer despite very few crossovers mapping to this region. This implicated this part of the acceptor in transfers occurring at downstream positions. For Env the results support a mechanism where the acceptor rapidly binds nascent DNA, then "zippers" downstream catching up with the donor-DNA hybrid and displacing the donor. Such a mechanism may be important to recombination in low structure regions of the HIV genome.
Derebail
Suchitra S
SS
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland 20742, USA.
DeStefano
Jeffrey J
JJ
D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015685:Fusion Proteins, gag-pol / Q000235:genetics*; D015752:Genes, env; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D004279:DNA, Viral; D015685:Fusion Proteins, gag-pol; D012367:RNA, Viral