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

Abstract

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

The DNA damage response promotes polyomavirus JC infection by nucleus to cytoplasm NF- kappaB activation

“…Cells were harvested as we have described [ 13 , 33 , 34 ] except for experiments to measure sumoylation where iodoacetemide (10 mM) was added to the lysis and wash buffers to preserve SUMOylation. Western blots were performed as described [ 33 , 35 , 36 ].…”

“…Blots were incubated with IRDye® 680RD Goat Anti-Mouse Li-COR dyes and visualized with an Odyssey® CLx Imaging System (LI-COR, Inc., Lincoln, NE) using LI-COR Odyssey software. Band intensities were quantified using the Quantity One software (Bio-Rad, Hercules CA) and intensities normalized to loading control as previously described [ 34 ].…”

“…Transfection of expression plasmids was performed using Lipofectamine 2000 (Life Technologies Inc., Carlsbad CA) was performed as we have previously described [ 33 , 34 ]. The total amount of transfected DNA was normalized with empty vector DNA.…”

“…TC620 cells were transfected with expression plasmids for FLAG-NEMO and JCV T-Ag, whole cell extracts prepared and IP/Western performed as we have described [ 33 , 34 ]. ATM was immunoprecipitated followed by Western blot for FLAG or vice versa for the reciprocal IP/Western.…”

The Brd4 acetyllysine-binding protein is involved in activation of polyomavirus JC

“…Blots were incubated with IRDye® 680RD Goat Anti-Mouse Li-COR dyes and visualized with an Odyssey® CLx Imaging System (LI-COR, Inc., Lincoln, NE) using LI-COR Odyssey software. Band intensities were quantified using the Quantity One software (Bio-Rad, Hercules CA) and intensities normalized to loading control as previously described (Bellizzi et al 2015). …”