Figure 2

MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1 during infection. (A) Cf2Th cells stably expressing FLAG-tagged MxB (MxB-FLAG), TRIM5α_rh or containing the empty vector LPCX were challenged using similar amounts of HIV-1. Sixteen hours post-infection samples were processed for the fate of the capsid assay as described in Methods. Input, soluble and pellet fractions were analyzed by Western blotting using anti-p24 antibodies (left panel). The binding of MxB to the viral core was assayed by Western blotting using anti-FLAG antibodies (left panel). The quantification of pelletable fractions relative to the pellet found when infecting cells containing the LPCX control vector by wild type HIV-1 was calculated for each experiment. Similar results were obtained in three independent experiments, and the standard deviations are shown. To control for the presence of MxB-FLAG in pelletable fractions when using mock-infected cells, Cf2Th cells stably expressing MxB-FLAG were challenged with HIV-1 or not, and processed for the fate of the capsid assay (right panel). Input and pellet fractions were analyzed by Western blotting using anti-p24 and anti-FLAG antibodies. Similar results were obtained in three independent experiments and a representative experiment is shown. (B) Cf2Th cells stably expressing MxB or stably transduced with the empty vector LPCX were challenged using similar amounts of HIV-1-P90A (left panel), HIV-1-G89V (middle panel) or HIV-1-N57S (right panel). Sixteen hours post-infection samples were processed for the fate of the capsid assay. Input, soluble and pellet fractions were analyzed by Western blotting using anti-p24 antibodies. The quantification of pelletable fractions relative to the pellet found when infecting cells containing the LPCX control vector by wild type HIV-1 was calculated for each experiment. Similar results were obtained in three independent experiments, and the standard deviations are shown. (C) Cf2Th cells stably expressing MxB, NES-CPSF6, TRIM5α_rh or containing the empty vector LPCX were challenged using similar amounts of HIV-1. Sixteen hours post-infection samples were processed for the fate of the capsid assay. Input, soluble and pellet fractions were analyzed by Western blotting using anti-p24 antibodies. Similar results were obtained in three independent experiments, and the standard deviation for pelletable fractions relative to the pellet found in cells containing the empty vector LPCX are shown. (D) IFN-α-treated (1000U/ml for 24 hours) or untreated Human HT1080 cells were challenged using similar amounts of HIV-1. Sixteen hours post-infection samples were processed for the fate of the capsid assay (left panel). Quantification of pelletable fractions relative to the pellet found when infecting untreated HT1080 cells by wild type HIV-1 was calculated for each experiment. Similar results were obtained in three independent experiments, and the standard deviations are shown. Induction of MxB expression in human HT1080 cells by the indicated amounts of IFN-α after 24 hours of exposure is shown (upper right panel). The ability of IFN-α-treated HT1080 cells to block HIV-1 infection is shown (bottom right panel). (E) MxB causes retention of HIV virions in the cytoplasm. Cf2Th cells stably expressing MxB, MxB-∆(1–20), or containing the empty vector LPCX, were spinoculated with fluorescently labeled HIV-1 virions for 2 h. HIV-1 viruses were fluorescently labeled with Vpr-GFP (label the inside of the virus) and the S15-mCherry (labels the lipid membrane of the virus). Cells were fixed and analyzed 3 and 6 hours post-infection. The total number of fused viruses (S15-mCherry negative particles) is shown, as determined by counting 10 fields using the Imaris software. On the left panel, the relative number of fused virions normalized to input viruses overtime are shown. On the right panel, the percentage of total fused virions retained in the cytoplasm overtime is shown. Results are representative of 3 independent experiments. ***, P <0.001 and **, P <0.05, as compared with respective controls.