TRAF6 but not VISA controls the level of expression of IRF7 and HIV replication.

Primary human macrophages were subjected to siRNA knockdown for TRAF6 and VISA. The concentration of siRNA used was 200 nM. A) Percentages of inhibition of TRAF6 and VISA in the presence of specific siRNA targeting both gene individually [n = 3] compared to a control non-targeted siRNA [n = 3]. Level of expression was measured by qRT-PCR B) Macrophages were all infected with HIV-1 with and without an IFNα2 pretreatment, and treated with specific siRNA. The level of TAT spliced message measured 24 hours post-infection in TRAF6 and VISA conditions were compared to the negative control siRNA. C) Level of IRF7 and D) Level of IRF3 message expression in the absence of IFNα2 when TRAF6 and VISA are repressed by siRNA compared to the negative control siRNA. The qRT-PCR results were normalized to the level of 18S. An * denotes a significant difference (P<0.05, one sample t-test) compared to the negative control siRNA. Each symbol in a group represents one independent donor. E) Western blot for IRF3 and IRF7, 24 hours post-infection in macrophages with TRAF6 suppression compared to siRNA control. The densitometry of the bands was normalized to that of β-actin and shows a decrease in protein level of IRF3 and an increase of IRF7. The ratio is shown next to the respective panels. Results shown are representative of 3 independent experiments.