FIG 5.

Vpr degrades TET2 to reduce demethylation of the IFITM3 promoter in MDMs. (A and B) TET2 is required for the constitutive expression of IFITM3 in MDMs. (A) Western blot analysis of cell lysates was performed with anti-TET2 or anti-IFITM3 antibody. Relative IFITM3 expression is calculated as IFITM3/actin ratios. (B) MDMs with control or TET2 shRNA were treated with VLPs with or without Vpr for 24 h. TET2 and IFITM3 mRNA levels were detected by RT-qPCR. (C and D) TET2 binds to the IFITM3 promoter to catalyze its demethylation. (C) MDMs transduced with control or TET2 shRNA were treated with VLPs with or without Vpr for 24 h. The ChIP assay was performed with anti-TET2 monoclonal antibody (MAb) to determine TET2 binding to the IFITM3 promoter. (D) ChIP with anti-5hmC MAb was performed to determine demethylation at the IFITM3 promoter. Specific IFITM3 promoter DNA by ChIP is normalized to the DNA ChIP with control IgG. DNA ChIP with control IgG is defined as 1. * and ** indicate P < 0.05 and P < 0.01, respectively. (E) Schematic diagram of TET2-mediated demethylation of the IFITM3 promoter.