Enteroviruses Manipulate the Unfolded Protein Response through Multifaceted Deregulation of the Ire1-Xbp1 Pathway

Abstract

Many viruses are known to trigger endoplasmic reticulum (ER) stress in host cells, which in turn can develop a protective unfolded protein response (UPR). Depending on the conditions, the UPR may lead to either cell survival or programmed cell death. One of three UPR branches involves the upregulation of Xbp1 transcription factor caused by the unconventional cytoplasmic splicing of its mRNA. This process is accomplished by the phosphorylated form of the endoribonuclease/protein kinase Ire1/ERN1. Here, we show that the phosphorylation of Ire1 is up-regulated in HeLa cells early in enterovirus infection but down-regulated at later stages. We also find that Ire1 is cleaved in poliovirus- and coxsackievirus-infected HeLa cells 4-6 h after infection. We further show that the Ire1-mediated Xbp1 mRNA splicing is repressed in infected cells in a time-dependent manner. Thus, our results demonstrate the ability of enteroviruses to actively modulate the Ire1-Xbp1 host defensive pathway by inducing phosphorylation and proteolytic cleavage of the ER stress sensor Ire1, as well as down-regulating its splicing activity. Inactivation of Ire1 could be a novel mode of the UPR manipulation employed by viruses to modify the ER stress response in the infected cells.

Keywords: ER stress; Ire1 proteolysis; Ire1-Xbp1 pathway; coxsackievirus; picornavirus; poliovirus; unfolded protein response.

Figure 1

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.

Figure 2

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.

Figure 3

Ire1 proteolytic cleavage in the PV-infected HeLa cells is not affected by the pan-caspase inhibitor Q-VD-Oph. HeLa cells were infected with PV in the absence or presence of 20 μM Q-VD-Oph. After the time indicated, cells were lysed and analyzed by Western blotting with polyclonal anti-Ire1 antibodies (Abcam #ab37073). Asterisks denote the same as in Figure 2.

Figure 4

Enterovirus infection does not induce Xbp1 mRNA splicing and activation of the Xbp1 downstream target ERdj4. (A) Total RNA from HeLa cells infected with PV, mock-infected, or 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) The same experiment with CVB3. Each experiment was performed in duplicate and repeated at least three times. (C) Total RNA from HeLa cells infected with PV, mock-infected, or treated with 10 mM DTT was isolated at the indicated time points. The relative level of ERdj4 mRNA was measured by RT-qPCR with primers specific to the ERdj4 mRNA and RPL19 mRNA as a reference. (D) The same experiment with CVB3.

Figure 5

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.

Figure 6

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.