title: Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors.
abstract: We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.
authors.affiliation: From the Institute for Molecular Virology.
authors.forename: Krishan K
authors.initials: KK
authors.lastname: Pandey
authors.affiliation: From the Institute for Molecular Virology.
authors.forename: Sibes
authors.initials: S
authors.lastname: Bera
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Sergey
authors.initials: S
authors.lastname: Korolev
authors.affiliation: Center for World Health and Medicine, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104 and.
authors.forename: Mary
authors.initials: M
authors.lastname: Campbell
authors.affiliation: the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Zhiqi
authors.initials: Z
authors.lastname: Yin
authors.affiliation: the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Hideki
authors.initials: H
authors.lastname: Aihara
authors.affiliation: From the Institute for Molecular Virology, grandgdp@slu.edu.
authors.forename: Duane P
authors.initials: DP
authors.lastname: Grandgenett
mesh_terms: D004279:DNA, Viral / Q000737:chemistry* / Q000276:immunology; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D017434:Protein Structure, Tertiary; D052736:Rous sarcoma virus / Q000737:chemistry* / Q000502:physiology; D019065:Virus Assembly / Q000502:physiology
chemical_list: D004279:DNA, Viral; D019427:HIV Integrase
keywords: AIDS; Alpharetrovirus; Antiviral Agent; DNA-Protein Interaction; Enzyme Inhibitor; HIV-1 Protease; Integrase