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. 2004 May;78(10):5045–5055. doi: 10.1128/JVI.78.10.5045-5055.2004

FIG. 7.

FIG. 7.

RT-IN interaction does not involve disulfide bond formation. (A) Effect of a reducing agent on RT-IN interaction. HIV-1 IN was incubated in the absence (−) (lanes 1 and 3) or presence (+) (lanes 2 and 4) of RT and immunoprecipitated with a rabbit anti-HIV-1 RT polyclonal antibody. The immunoprecipitated complex was left untreated (lanes 1 and 2) or treated with β-ME (lanes 3 and 4), separated by SDS-PAGE, and analyzed by Western blotting using the rabbit anti-IN polyclonal antibody. The 64-kDa band in lane 2 corresponds to IN homodimers (5). The lower level of IN in lane 4 compared to lane 2 is due to a direct effect of β-ME on IN, possibly by altering the reactivity of IN to the anti-IN antibody. IgG-γ, immunoglobulin G heavy chain. (B) Effect of an alkylating agent on RT-IN interaction. Forty picomoles of HIV-1 IN in a final volume of 10 μl was preincubated at room temperature for 15 min in buffer C (20 mM HEPES-Na, pH 6.8, 250 mM NaCl, 0.2 mM MgCl2) with (lanes 1 and 2) or without (lanes 3 to 6) 10 mM DTT (20 mM equivalent of free sulfhydryls). The IN was then treated with 3.6 (2× molar excess; lanes 3 and 4) or 18 (10× molar excess; lanes 1, 2, 5, and 6) mM NEM for 1 h at 4°C. In lanes 3 to 6, the activity of NEM was quenched with 10 mM DTT. The samples were then added to buffer C with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) RT. Coimmunoprecipitation was carried out with the rabbit anti-HIV-1 RT polyclonal antibody. The immunoprecipitated complex was eluted with the buffer containing β-ME, and the rest of the procedure was as described for panel A. The extent of NEM modification was monitored using electrospray ionization-mass spectrometry. At 2× molar excess of NEM, all INs were modified, and the distribution of INs containing four, five, and six NEM adducts was ∼35, 50, and 10%, respectively (data not shown).The IN with four NEM adducts likely represented Cys modification at positions 56, 65, 130, and 280, since the Cys residues at positions 40 and 43 are bound with zinc and therefore should be much less reactive. The extent of NEM adduct formation at 10× excess was not determined.