Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1.

The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated degradation (ERAD) are two essential components of the quality control system for proteins in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an ERAD substrate, indicating that the UPR and ERAD regulate each other. Viruses are intracellular parasites that exploit the host cell for their own benefit. Cytomegaloviruses selectively modulate the UPR to take advantage of beneficial and inhibit detrimental effects on viral replication. We have previously shown that murine and human cytomegaloviruses express homologous proteins (M50 and UL50, respectively) that dampen the UPR at late times post infection by inducing IRE1 degradation. However, the degradation mechanism has remained uncertain. Here we show that the cytomegalovirus M50 protein mediates IRE1 degradation by the proteasome. M50-dependent IRE1 degradation can be blocked by pharmacological inhibition of p97/VCP or by genetic ablation of SEL1L, both of which are components of the ERAD machinery. SEL1L acts as a cofactor of the E3 ubiquitin ligase HRD1, while p97/VCP is responsible for the extraction of ubiquitylated proteins from the ER to the cytosol. We further show that M50 facilitates the IRE1-SEL1L interaction by binding to both, IRE1 and SEL1L. These results indicate that the viral M50 protein dampens the UPR by tethering IRE1 to SEL1L, thereby promoting its degradation by the ERAD machinery.IMPORTANCE Viruses infect cells of their host and force them to produce virus progeny. This can impose stress on the host cell and activate counter-regulatory mechanisms. Protein overload in the endoplasmic reticulum (ER) leads to ER stress and triggers the unfolded protein response, which in turn upregulates protein folding and increases the degradation of proteins in the ER. Previous work has shown that cytomegaloviruses interfere with the unfolded protein response by degrading the sensor molecule IRE1. Herein we demonstrate how the cytomegalovirus M50 protein exploits the ER-associated degradation machinery to dispose of IRE1. Degradation of IRE1 curbs the unfolded protein response and helps the virus to increase the synthesis of its own proteins and the production of virus progeny.

Cytomegalovirus downregulates IRE1 to repress the unfolded protein response.

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.

Repression of viral gene expression and replication by the unfolded protein response effector XBP1u.

The unfolded protein response (UPR) is a cellular homeostatic circuit regulating protein synthesis and processing in the ER by three ER-to-nucleus signaling pathways. One pathway is triggered by the inositol-requiring enzyme 1 (IRE1), which splices the X-box binding protein 1 (Xbp1) mRNA, thereby enabling expression of XBP1s. Another UPR pathway activates the activating transcription factor 6 (ATF6). Here we show that murine cytomegalovirus (MCMV), a prototypic ß-herpesvirus, harnesses the UPR to regulate its own life cycle. MCMV activates the IRE1-XBP1 pathway early post infection to relieve repression by XBP1u, the product of the unspliced Xbp1 mRNA. XBP1u inhibits viral gene expression and replication by blocking the activation of the viral major immediate-early promoter by XBP1s and ATF6. These findings reveal a redundant function of XBP1s and ATF6 as activators of the viral life cycle, and an unexpected role of XBP1u as a potent repressor of both XBP1s and ATF6-mediated activation.

Cells survive by making many different proteins that each carry out specific tasks. To work correctly, each protein must be made and then folded into the right shape. Cells carefully monitor protein folding because unfolded proteins can compromise their viability. A protein called XBP1 is important in controlling how cells respond to unfolded proteins. Normally, cells contain a form of this protein called XBP1u, while increasing numbers of unfolded proteins trigger production of a form called XBP1s. The change from one form to the other is activated by a protein called IRE1. Viruses often manipulate stress responses like the unfolded protein response to help take control of the cell and produce more copies of the virus. Murine cytomegalovirus, which is known as MCMV for short, is a herpes-like virus that infects mice; it stops IRE1 activation and XBP1s production during the later stages of infection. However, research had shown that the unfolded protein response was triggered for a short time at an early stage of infection with MCMV, and it was unclear why this might be. Hinte et al. studied the effect of MCMV on cells grown in the laboratory. The experiments showed that a small dose of cell stress, namely activating the unfolded protein response briefly during early infection, helps to activate genes from the virus that allow it to take over the cell. Together, XBP1s and another protein called ATF6 help to switch on the viral genes. The virus also triggers IRE1 helping to reduce the levels of XBP1u, which could slow down the infection. Later, suppressing the unfolded protein response allows copies of the virus to be made faster to help spread the infection. These findings reveal new details of how viruses precisely manipulate their host cells at different stages of infection. These insights could lead to new ways to manage or prevent viral infections.