Degradation of polyomavirus JC T-antigen by stress involves the LIP isoform of C/EBP

Abstract

Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum. CCAAT/enhancer binding proteins are one of the cellular proteins whose expression is upregulated during ER stress. Previously, we have identified C/EBPbeta isoforms, especially LIP, as a negative regulator of polyomavirus JC (JCV), the causative agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Here, we show that the induction of ER stress by thapsigargin increase the expression of endogenous LIP and the degradation of JCV T-antigen in a JCV-transgenic mouse tumor cell line. Our results also revealed that overexpression of LIP significantly reduced the level of T-Ag and this effect is reversed upon siRNA-mediated silencing of LIP. Immunoprecipitation/Western blot experiments indicated that LIP interacts with T-antigen directly. Treatment of cells that overexpress LIP with MG115, a proteasome inhibitor, partially rescued LIP-mediated degradation of T-antigen. Our observations point to a role of LIP in ER stress regulation of T-antigen stability and may open a new avenue to study host-virus interaction during ER stress.

Keywords: CAAT/enhancer binding protein-beta; endoplasmic reticulum stress; large transforming antigen; liver-inhibitory isoform; polyomavirus JC.

Figure 1.

Effect of thapsigargin on the expression of LIP and T-Ag. BsB8 cells were treated with thapsigargin (TG) and then analyzed by Western blot for expression of T-Ag (A) and LIP (B) as described in Materials and Methods. The loading control was α-tubulin. The left-hand lane contains the molecular weight markers (MW). The effect of TG on BsB8 cell viability was measured by MTT assay (C).

Figure 2.

Effect of overexpression of LIP and LIP siRNA on T-Ag expression. (A) BsB8 cells were transfected with different amounts of expression plasmid for LIP as indicated and T-Ag expression analyzed by Western blot. The loading control was α-tubulin. The left-hand lane contains the molecular weight markers (MW). (B) BsB8 cells were transfected with expression plasmid for LIP and transduced with adenovirus vectors as indicated and T-Ag expression analyzed by Western blot. The loading control was α-tubulin. The left-hand lane contains the molecular weight markers (MW).

Figure 3.

Effect of MG118 on LIP downregulation of T-Ag expression. BsB8 cells were transfected with different amounts of expression plasmid for LIP in the absence and presence of MG115 as indicated and T-Ag expression analyzed by Western blot. The loading control was α-tubulin.

Figure 4.

Immunoprecipitation(IP)/Western blot of LIP and T-Ag. BsB8 cells were transfected with expression plasmid for LIP, whole cell extract (WCE) prepared and: (A) IP performed with antibody to T-Ag or nonimmune mouse serum (NMS) followed by Western blot for LIP and T-Ag. The left-hand lane contains the molecular weight markers (MW). (B) IP performed with antibody to LIP or nonimmune mouse serum (NMS) followed by Western blot for LIP and T-Ag. The left-hand lane contains the molecular weight markers (MW).