Figure 3

MLN4924 inhibits Vif function. HEK293T cells were transfected with an expression vector for APOBEC3G–HA, and either empty vector, pNL-A1, or pNL-A1Δvif. Twenty four hours later, the cells were either mock treated or treated overnight with 1 μM MLN4924. Cells were then harvested and immunoblotted for the HA epitope, β-actin, or HIV-1 p24 (A). HEK293T cells were co-transfected with an expression vector for APOBEC3G-HA and either an expression vector for HIV-1 or one for a Δ vif HIV-1 along with an expression vector for the vesicular stomatitis virus glycoprotein (VSV-G). Twenty four hours later, cells were either mock treated or treated with 1 μM MLN4924, and cultured overnight. Virus-containing supernatants were collected and immunoblotted for HIV-1 p24 and the HA epitope (B). HEK293T cells were co-transfected with HIV-1 or Δvif HIV-1 constructs along with an expression vector for VSV-G and either empty vector or APOBEC3G–HA. Twenty four hours later, cells were either mock treated or treated with 1 μM MLN4924, and left overnight. The virus-containing supernatants were collected and used to infect GHOST cells. Twenty four hours post infection; the cells were harvested, fixed, and analyzed for GFP expression by flow cytometry (C). Panel D summarizes data from multiple experiments performed as indicated for panel C and error bars show standard error. The two-tailed Student’s t-test was used to determine whether differences between pairs of conditions are statistically significant. ns indicates comparisons where the differences were not significant at a level of 0.05.