title: HIV-1 nucleocapsid protein increases strand transfer recombination by promoting dimeric G-quartet formation.
abstract: A preferred site for HIV-1 recombination was identified in vivo and in vitro surrounding the beginning of the HIV-1 gag gene. This G-rich gag hotspot for recombination contains three evenly spaced G-runs that stalled reverse transcriptase. Disruption of the G-runs suppressed both the associated pausing and strand transfer in vitro. Significantly, this same gag sequence was able to fold into a G-quartet monomer, dimer, and tetramer, depending on the cations employed. The pause band at the G-run (nucleotide (nt) 405-409), which was predicted to be involved in forming a G-quartet monomer, diminished with increased HIV-1 nucleocapsid (NC) protein. More NC induced stronger pauses at other G-runs (nt 363-367 and nt 382-384), a region that forms a G-quartet dimer, adhering the two RNA templates. We hypothesized that NC induces the unfolding of the monomeric G-quartet but stabilizes the dimeric interaction. We tested this by inserting a known G-quartet formation sequence, 5'-(UGGGGU)(4)-3', into a relatively structure-free template from the HIV-1 pol gene. Strand transfer assays were performed with cations that either encourage (K(+)) or discourage (Li(+)) G-quartet formation with or without NC. Strikingly, a G-quartet monomer was observed without NC, whereas a G-quartet dimer was observed with NC, both only in the presence of K(+). Moreover, the transfer efficiency of the dimerized template (with K(+) and NC) reached about 90%, approximately 2.5-fold of that of the non-dimerized template. Evidently, template dimerization induced by NC creates a proximity effect, leading to the unique high peak of transfer at the gag recombination hotspot.
authors: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors: Wen
authors: W
authors: Shen
authors: Robert J
authors: RJ
authors: Gorelick
authors: Robert A
authors: RA
authors: Bambara
mesh_terms: D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000502:physiology*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase