RNA motifs mediating in vivo site-specific nonhomologous recombination in (+) RNA virus enforce in vitro nonhomologous crossovers with HIV-1 reverse transcriptase.
There are several lines of evidence that both RNA viruses and retroviruses recombine according to a copy choice mechanism. Using the brome mosaic virus (BMV)-based system, we recognized elements in the RNA structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. The same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to switch templates during DNA synthesis. We demonstrated that a specific combination of the local double-stranded region with short homologous sequences and a hairpin structure allows template switching by HIV-RT. In contrast to BMV replicase, HIV-RT does not mediate the detectable level of recombination using only the heteroduplex structure, though local hybridization between RNA molecules efficiently pauses primer extension. Moreover, the presented data suggest that a proper arrangement of identified structural motifs can ensure site specificity of RNA-RNA recombination. These results indicate that HIV-RT utilizes the same or a very similar mechanism as BMV replicase to change nonhomologous RNA templates in a site-specific manner.
Figlerowicz
M
M
Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań. marekf@ibch.poznan.pl
Bibiłło
A
A
D001483:Base Sequence; D017795:Bromovirus / Q000201:enzymology* / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000201:enzymology* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic
D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase