title: Relative rates of retroviral reverse transcriptase template switching during RNA- and DNA-dependent DNA synthesis.
abstract: Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site (att) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and att site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.
authors.affiliation: Department of Genetics and Developmental Biology, West Virginia University, Morgantown, West Virginia 26506, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Bowman
authors.forename: W S
authors.initials: WS
authors.lastname: Hu
authors.forename: V K
authors.initials: VK
authors.lastname: Pathak
mesh_terms: D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D012367:RNA, Viral / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000201:enzymology* / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D004247:DNA; D012194:RNA-Directed DNA Polymerase