Acceptor RNA cleavage profile supports an invasion mechanism for HIV-1 minus strand transfer.
We previously proposed that HIV-1 minus strand transfer occurs by an acceptor invasion-initiated multi-step mechanism. During synthesis of minus strong stop DNA, reverse transcriptase (RT) transiently pauses at the base of TAR before continuing synthesis. Pausing promotes RT-RNase H cleavage of the donor RNA, exposing regions of the cDNA. The acceptor RNA then invades at these locations to interact with the minus strong stop DNA. Whereas primer extension continues on the donor RNA, the cDNA-acceptor hybrid expands by branch migration until transfer of the primer terminus is completed. We present results here showing that the interaction of the acceptor RNA and the cDNA can be determined by examining the time-dependent cleavage of the acceptor RNA by RNase H. Our approach utilizes a combination of RT-RNase H and Escherichia coli RNase H to allow assessment of acceptor-cDNA interactions at high sensitivity. Results show an initial interaction of the acceptor RNA with cDNA at the base of TAR. We observe a time-dependent shift in RNase H susceptibility along the length of the acceptor toward the 5' end, suggesting hybrid propagation from the initial invasion point. Control experiments validate that the RNase H cleavage profile represents the formation and expansion of the acceptor-DNA interaction and that the process is promoted by the nucleocapsid. Observations with this new approach lend additional support to the proposed multistep transfer mechanism.
Chen
Yan
Y
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
Balakrishnan
Mini
M
Roques
Bernard P
BP
Bambara
Robert A
RA
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D008957:Models, Genetic; D019251:Nucleocapsid / Q000737:chemistry; D019590:Nucleocapsid Proteins / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D012260:Ribonucleases; D016914:Ribonuclease H