CSFV Infection Up-Regulates the Unfolded Protein Response to Promote Its Replication
Jinding Chen, Mingqiu Zhao
Classical swine fever (CSF) is an OIE-listed, highly contagious animal disease caused by classical swine fever virus (CSFV). The endoplasmic reticulum (ER) is an organelle in which the replication of many RNA viruses takes place. During viral infection, a series of events elicited in cells can destroy the ER homeostasis that cause ER stress and induce an unfolded protein response (UPR). In this study, we demonstrate that ER stress was induced during CSFV infection as several UPR-responsive elements such as XBP1(s), GRP78 and CHOP were up-regulated. Specifically, CSFV transiently activated IRE1 pathway at the initial stage of infection but rapidly switched off, likely due to the reduction in cytoplasm Ca2+ after viral incubation. Additionally, our data show that the ER stress induced by CSFV can promote CSFV production, which the IRE1 pathway play an important role in it. Evidence of ER stress in vivo was also confirmed by the marked elevation of GRP78 in CSFV-infected pig PBMC and tissues. Collectively, these data indicate that the ER stress was induced upon CSFV infection and that the activation of the IRE1 pathway benefits CSFV replication.The primary antibodies used in the study were specific for GRP78 (Santa Cruz Biotechnology, sc-13968), GRP94 (Novus, NBP2-44690-0.02mg), eIF2α (phospho-S51) (Bioworld, BS4787), eIF2α (Bioworld, BS3651), IRE1 (phosphor S724) (Abcam, ab48187), IRE1 (Bioss, bs-8680R), ATF6 (ABclonal, A0202), ATF4 (ImmunoWay, YT1102), CHOP (Santa Cruz Biotechnology, sc-166682), CSFV-E2 (JBT, 2229), CSFV-Npro (a gift from Hunan agricultural university professor YuXingLong), Tubulin (Beyotime, AT819), and Actin (Beyotime, AT0003). The secondary antibodies Goat anti-Mouse IgG-HRP (Bioworld, BS12478) and Goat anti-Rabbit IgG-HRP (Bioworld, BS13278) were purchased from Bioworld Technology. Thapsigargin (Calbiochem®, 67526-95-8), 4μ8c (selleck.cn, S7272) and BIX (Sigma, SML1073) were dissolved in dimethyl sulfoxide (DMSO) at the indicated concentrations. Tauroursodeoxycholic acid (sodium salt, Merck-Millipore, 580549-1GM) and NH4CL (Damao Chemical Reagent, 12125-02-9) were dissolved in ddH2O at the indicated concentrations. Fluo4-am (BestBio, BB-48113) was diluted 50-folds with Hank’s Balanced Salt Solution (HBSS). pEGFP-N1 and pEGFP-GRP78 were prepared in our laboratory. Additionally, shRNA vector targeting the coding sequences of GRP78 and the scrambled shRNA were designed by and purchased from Cyagen.This work was supported by grants from the National Natural Science Foundation of China (Nos. U1405216, 31472200 and 31672590), the Science and Technology Program of Guangzhou, China (Nos. 2015A050502044 and 2015B020230009) and National Key Research and Development Program (Nos. 2016YFD0500707 and 2016YFD0500801).