Fig 2: A model explaining the modulation of the Ire1-Xbp1 pathway in enterovirus-infected cells by the induction of Ire1 phosphorylation and inactivation through multiple mechanisms, including proteolytic cleavage. The activated Ire1-Xbp1 pathway in uninfected (left) or enterovirus-infected (right) cells is shown.

Fig 3: Enteroviruses inhibit Ire1-mediated Xbp1 mRNA splicing at the middle, but not early stage of infection. (A) HeLa cells were infected with PV or CVB3, then at 3 hpi 10 mM DTT was added to the medium. 2 h later, total RNA was isolated and the relative levels of the spliced Xbp1 mRNA were measured by RT-qPCR, as described earlier, ** p < 0.01. (B) Relative Xbp1s mRNA level in PV-infected HeLa cells with and without the addition of 10 mM DTT at 0 hpi, as revealed by RT-qPCR. (C) The same experiment as in (B), with CVB3. Each experiment was performed in duplicate and repeated at least three times. (D) DTT-induced Xbp1 mRNA splicing in PV-infected cells analyzed by agarose gel electrophoresis of RT-PCR products. HeLa cells were infected with PV and treated with 10 mM DTT at the indicated time points. 2 h later, cells were harvested, RNA was extracted, and 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. Note that the amounts of the two PCR products should not be compared to each other, as they have different lengths.

Fig 4: Ire1 phosphorylation in enterovirus-infected cells as revealed by Western blot analysis with anti-p-Ire1 antibodies. (A) Phosphorylated Ire1 and total β-actin levels in HeLa cells infected with PV type I Mahoney. (B) The same for HeLa cells infected with CVB3. Representative results of at least three independent experiments are shown. (C) Accumulation of viral proteins throughout the infection cycle of PV and CVB3 in HeLa cells.

Fig 5: Total Ire1 protein levels in picornavirus-infected HeLa cells analyzed on Western blots using different antibodies. (A) Ire1 levels in PV-infected HeLa cells visualized using anti-Ire1 polyclonal antibodies from Sigma (#I6785). (B) Ire1 levels in CVB-infected HeLa cells visualized using the same antibodies (#I6785). (C) Ire1 levels in PV-infected HeLa cells visualized using an anti-Ire1 polyclonal antibody from Abcam (#ab37073). (D) Ire1 levels in EMCV-infected HeLa cells visualized on Western blots using antibodies from Abcam. Representative results of at least three independent experiments are shown. Ire1-FL—the full-length Ire1; Ire1-Cl—products of Ire1 proteolytic cleavage; *—non-specific signals.