Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

• Published in: The Journal of biological chemistry 2014-07, Vol.289 (28), p.19648-19658
• Main Authors: Pandey, Krishan K., Bera, Sibes, Korolev, Sergey, Campbell, Mary, Yin, Zhiqi, Aihara, Hideki, Grandgenett, Duane P.
• Format: Article
• Language: English
• Subjects: AIDS; Alpharetrovirus; Antiviral Agent; DNA, Viral - chemistry; DNA, Viral - immunology; DNA-Protein Interaction; Enzyme Inhibitor; Enzymology; HIV Integrase - chemistry; HIV Integrase - metabolism; HIV-1 - chemistry; HIV-1 - physiology; HIV-1 Protease; Humans; Integrase; Protein Structure, Tertiary; Rous sarcoma virus - chemistry; Rous sarcoma virus - physiology; Virus Assembly - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.573311

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. Background: Structure of the three-domain Rous sarcoma virus integrase with viral DNA is lacking. Results: Soluble (>1.5 mg/ml) and stable synaptic complex using Rous sarcoma virus integrase, DNA, and HIV-1 strand transfer inhibitors was produced. Conclusion: Two integrase dimers assemble onto viral DNA to produce a synaptic complex. Significance: This is the first report producing a high concentration of soluble and kinetically stabilized RSV synaptic complex.