Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase.
Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.
Hanson
Mark Nils
MN
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Balakrishnan
Mini
M
Roques
Bernard P
BP
Bambara
Robert A
RA
D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019251:Nucleocapsid / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000378:metabolism; D013698:Templates, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H