FIG 8.
LANA interferes with the association of CIITA and RFXAP. (A) Coimmunoprecipitation assay showing reduced associations of RFXAP with CIITA in the presence of LANA. Flag-tagged CIITA, Myc-tagged RFXAP, and Myc-tagged LANA-N (aa 1 to 340) were used in this competitive coimmunoprecipitation assays after transfection into HEK293T cells. CIITA was immunoprecipitated from the cellular lysates by using an anti-Flag antibody. The proteins were resolved on SDS-PAGE gels, and coimmunoprecipitated proteins were detected by using anti-Myc and anti-Flag antibodies. HC, heavy chain. (B) Coimmunoprecipitation assay showing reduced association of RFX5 with CIITA in an in vitro binding assay. In vitro-translated and [35S]methionine-labeled LANA-N (aa 1 to 340)–Myc, RFXAP-Myc, RFX5-Myc, RFXANK-Myc, and CIITA-Flag were used for in vitro binding assays. The proteins were allowed to interact overnight at 4°C, followed by immunoprecipitation with anti-Flag antibody (CIITA). The bound protein complexes were resolved on SDS-PAGE gels, followed by detection by autoradiography. (Ca) ChIP assays showing reduced binding of CIITA to the HLA-DRA promoter in LANA-expressing BJAB cells compared to control YFP-expressing cells. (b and c) Relative binding of CIITA in LANA-depleted and control KSHV-infected BC-3 (b) and BCBL-1 (c) PEL cells. (d) Relative binding of CIITA on the chromatin HLA-DRA promoters in the expression system. The HLA-DRA-Luc vector and RFX components (Myc tagged) were transfected with the Flag vector (control), CIITA-Flag, or CIITA-Flag with LANA-Myc in HEK293L cells, followed by a ChIP assay with anti-Flag (CIITA) antibody. The amount of CIITA-bound HLA-DR was reduced in LANA-expressing cells. The error bars represent standard deviations of the means from at least three experimental replicates. P values were calculated by two-tailed t tests comparing treated to control cells. ***, P < 0.001; ****, P < 0.0001. (D) Immunoblot showing the expression of LANA and CIITA and the immunoprecipitation efficiency of anti-CIITA antibody used for ChIP assays. (a) LANA-expressing (LYF) and control YFP (YF) BJAB cells. (b and c) LANA depletion in KSHV-infected BC-3 (b) and BCBL-1 (c) PEL cells by the lentiviral vector showing efficient knockdown of LANA compared to shControl cells. (d) Immune detection of LANA and CIITA in inputs and immunoprecipitated cells with anti-Myc and anti-Flag antibodies, respectively.