title: A recombination hot spot in HIV-1 contains guanosine runs that can form a G-quartet structure and promote strand transfer in vitro.
abstract: The co-packaged RNA genomes of human immunodeficiency virus-1 recombine at a high rate. Recombination can mix mutations to generate viruses that escape immune response. A cell-culture-based system was designed previously to map recombination events in a 459-bp region spanning the primer binding site through a portion of the gag protein coding region. Strikingly, a strong preferential site for recombination in vivo was identified within a 112-nucleotide-long region near the beginning of gag. Strand transfer assays in vitro revealed that three pause bands in the gag hot spot each corresponded to a run of guanosine (G) residues. Pausing of reverse transcriptase is known to promote recombination by strand transfer both in vivo and in vitro. To assess the significance of the G runs, we altered them by base substitutions. Disruption of the G runs eliminated both the associated pausing and strand transfer. Some G-rich sequences can develop G-quartet structures, which were first proposed to form in telomeric DNA. G-quartet structure formation is highly dependent on the presence of specific cations. Incubation in cations discouraging G-quartets altered gel mobility of the gag template consistent with breakdown of G-quartet structure. The same cations faded G-run pauses but did not affect pauses caused by hairpins, indicating that quartet structure causes pausing. Moreover, gel analysis with cations favoring G-quartet structure indicated no structure in mutated templates. Overall, results point to reverse transcriptase pausing at G runs that can form quartets as a unique feature of the gag recombination hot spot.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Shen
authors.forename: Lu
authors.initials: L
authors.lastname: Gao
authors.forename: Mini
authors.initials: M
authors.lastname: Balakrishnan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D001483:Base Sequence; D002412:Cations; D016679:Genome, Viral; D006151:Guanosine / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics; D016662:Virus Integration; D014779:Virus Replication / Q000235:genetics*
chemical_list: D002412:Cations; D012367:RNA, Viral; D014764:Viral Proteins; D006151:Guanosine; D054303:HIV Reverse Transcriptase