Figure 1

MxB binds in vitro assembled HIV-1 CA-NC complexes. (A) The ability of MxB to bind in vitro assembled HIV-1 CA-NC complexes was measured as described in Methods. Input and Bound fractions were analyzed by Western blotting using anti-HA or anti-p24 antibodies. Similar results were obtained in three independent experiments, and the standard deviation for the bound fraction relative to input is shown. (B) The ability of MxB to bind in vitro assembled HIV-1 CA-NC complexes’ bearing the capsid changes P90A, G89V and N57S was measured as described above. Similar results were obtained in three independent experiments. (C) The ability of MxA, MxB and the protein chimera MxB (1–90)-MxA (43–662) to bind in vitro assembled HIV-1 CA-NC were measured as described above (left panel). Cf2Th cells stably expressing MxA, MxB, or MxB (1–90)-MxA (43–662) were challenged with increasing amounts of HIV-1-GFP viruses. Infection was determined by measuring the percentage of GFP-positive cells (right panel). MxA, MxB and MxB (1–90)-MxA (43–662) proteins are depicted (lower panel). Similar results were obtained in three independent experiments. NLS: nuclear localization signal. B: comprises the bundle signaling element. G: contains a GTPase domain. Stalk: domain that mediates assembly into oligomers. (D) Increased HIV-1 capsid colocalization with MxB upon infection. CF2Th cells stably expressing MxB, MxB-∆(1–20), or containing the empty vector LPCX, were spinoculated with VSV-G pseudotyped R7ΔEnv virions for 2 h. Cells were fixed 3 and 6 hours post-infection. Colocalization of MxB with HIV capsid (p24) is shown 6 hours post infection. The percentage of p24 associated with MxB was quantified by counting the number of p24 positive virions, which are positive for MxB is shown. Between 20–25 images were analyzed per condition using the Imaris Software as described in Methods. Bafilomycin A1 (Baf1). Data is representative of 3 independent experiments. **, P <0.05.