EC5S Ubiquitin Complex Is Recruited by KSHV Latent Antigen LANA for Degradation of the VHL and p53 Tumor Suppressors
Figure 5
The Tumor Suppressors p53 and VHL Are Inhibited in the KSHV Latently Infected and LANA-Expressing Cells
(A) IB analysis of KSHV-positive cell lines (BC-3, BCBL-1, JSC-1, and BC-1) and KSHV-negative cell lines (BJAB, DG-75, and Louckes) against p53, VHL, and β-actin. Ten million of each cell line were lysed and coimmunoprecipitated using anti-VHL antibodies followed by detection with anti-VHL. Cell lysate (5%) was used in IB assays with anti-p53 and anti–β-actin. The data show that p53 and VHL in KSHV-positive cells are more strongly inhibited than in KSHV-negative cells.
(B) The levels of p53 and VHL protein in B-lymphoma cells with KSHV latent infection. Ten million BJAB- or KSHV-infected BJAB (k-BJAB) cells were lysed and coimmunoprecipitated using anti-VHL antibodies followed by detection with anti-VHL. Cell lysate (5%) was used in IB assays against LANA, p53, and β-actin. The data showed that the tumor suppressor proteins p53 and VHL were inhibited during KSHV infection.
(C) p53 and VHL are inhibited in the LANA stable expressing cells. Ten million 293/LANA or 293/Vector stable cell lysates were analyzed by IB against LANA, p53, and VHL as described previously. The data showed that LANA decreases tumor suppressor p53 and VHL levels.
(D) p53 and VHL turnover in the LANA knockdown BCBL-1 cells. Cell lysates from stable BCBL-1 cells with LANA or control firefly luciferase knockdown (RNAi) were subjected to IB assays (left panel) against LANA, p53, VHL, and β-actin, or total RNA underwent RT-PCR analysis (right panel). RD, relative density.