Figure 3

Association of wild type and mutated HIV-1 _NL4–3 capsid with MxB. Wild type NL4-3 or its mutants V86A, V86Q, H87P, H87Q, A88V and A92P were used to infect MxB-expressing SupT1 cells or control SupT1 cells without MxB expression. At 16 hours after infection, cells were harvested, suspended in a hypotonic buffer containing 10 mM Tris–HCl (pH8.0), 10 mM KCl and 1 mM EDTA, and lysed with 15 strokes in a 7 ml Dounce homogenizer. After clearing cell debris by centrifugation at 3,000 rpm for 5 min at 4°C, cell lysates were incubated with the anti-FLAG M2 agarose (Sigma) for 4 hours at 4°C. After extensive washing, the bound MxB was eluted with 3xFLAG peptide (Sigma) and the amounts of associated HIV-1 p24 were determined by ELISA. (A) Levels of MxB-FLAG, wild type and the mutated HIV-1 p24/capsid in the infected SupT1 cells as determined by Western blotting. Amounts of wild type and mutated p24 were also quantified by ELISA and results are shown in the bar graph. (B) Levels of wild type and mutated p24 that were associated with MxB. Levels of the eluted MxB-FLAG were assessed by Western blotting. For each virus, the amounts of viral p24 eluted from the M2 agarose were determined by ELISA, then calibrated by p24 amounts in the corresponding cell lysates. Levels of MxB-associated p24 were determined by dividing the values of M2 agarose-associated p24 from MxB-expressing cells with those of M2 agarose-associated p24 from the control cells. Results shown are the average of three independent infection experiments. (C) Levels of viral p24 in NL4-3 infected SupT1 cells under treatment of CSA (5 μM). (D) Effect of CSA treatment on p24 association with MxB. Detailed legend refers to (B).