Evidence that creation of invasion sites determines the rate of strand transfer mediated by HIV-1 reverse transcriptase.
Strand transfer during reverse transcription can produce genetic recombination in human immunodeficiency virus type 1 (HIV-1) when two genomic RNAs, that are not identical, are co-packaged in the virus. Strand transfer was measured in vitro, in reactions involving primer switching from a donor to acceptor RNA template. The transfer product appeared with much slower kinetics than full-length synthesis on the donor template. The goal of this study was to learn more about the transfer mechanism by defining the steps that limit its rate. We previously proposed transfer to include the steps of acceptor invasion, hybrid propagation, terminus transfer, and re-initiation of synthesis on the acceptor template. Unexpectedly, with our templates increasing acceptor concentration increased the transfer efficiency but had no effect on the rate of transfer. Templates with a short region of homology limiting hybrid propagation exhibited a slow accumulation of transfer products, suggesting that for tested long homology templates hybrid propagation was not rate limiting. Substituting a DNA acceptor and adding Klenow polymerase accelerated re-initiation and extension exclusively on the DNA acceptor. This lead to a small rate increase due to faster extension on the acceptor, suggesting re-initiation of synthesis on the tested RNA acceptors was not rate limiting. A substrate was designed in which the 5' end of the primer was single stranded, and complimentary to the acceptor, i.e. having a pre-made invasion site. With this substrate, increasing concentrations of acceptor increased the rate of transfer. Together these data suggest that RNase H cleavage, and dissociation of RNA fragments creating an invasion site was rate limiting on most tested templates. When an accessible invasion site was present, acceptor interaction at that site influence the rate.
Hanson
Mark Nils
MN
Department of Biochemistry and Biophysics, Center, University of Rochester Medical Center, Rochester, NY 14642, USA.
Balakrishnan
Mini
M
Roques
Bernard P
BP
Bambara
Robert A
RA
D017931:DNA Primers; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000737:chemistry; D011995:Recombination, Genetic; D048348:Reverse Transcription; D013698:Templates, Genetic
D017931:DNA Primers; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase