Modulation of Ire1-Xbp1 Defense Pathway in Encephalomyocarditis Virus-Infected HeLa Cells

Figure 1. IRE1 phosphorylation in EMCV (Mengo strain)-infected HeLa cells. (A) Phosphorylated IRE1 and total β-actin levels in HeLa cells infected with EMCV Mengo strain. (B) Quantified normalized signal intensity of phosphorylated IRE1 to β-actin, relative to mock-infected cells. Representative results of at least three independent experiments are shown. * p < 0.05, ** p < 0.01.

Figure 2. IRE1 phosphorylation in EMCV (Mengo strain) and its mutant infected HeLa cells.

Figure 3. EMCV modulates XBP1 mRNA splicing in infected HeLa cells. (A) Total RNA from HeLa cells infected with wild-type EMCV and its mutants; mock-infected; and treated with 10 mM DTT was isolated at the indicated time points. The relative level of the spliced Xbp1 mRNA was measured by RT-qPCR with primers specific to Xbp1s mRNA isoform and to RPL19 mRNA as a reference. (B) HeLa cells were infected with EMCV and its mutants, then at 3 hpi, 10 mM DTT was added to the medium. Two hours later, total RNA was isolated and the relative levels of the spliced Xbp1 mRNA were measured by RT-qPCR. (C) The same experiment as in (A), RT-PCR with an Xbp1-specific primer pair producing fragments of either 442 or 416 bp (corresponding to the unspliced or spliced Xbp1 mRNAs, respectively), was performed, and fragments were analyzed on 2% agarose gel. (D) The same experiment as in (B), where fragments corresponding to unspliced and spliced form of XBP1 mRNA were analyzed on 2% agarose gel, was performed.