The Human Cytomegalovirus Endoplasmic Reticulum-Resident Glycoprotein UL148 Activates the Unfolded Protein Response.

Eukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER-resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol-requiring enzyme-1 (IRE1), as indicated by splicing of Xbp-1 mRNA, and the protein kinase R (PKR)-like ER kinase (PERK), as indicated by phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and accumulation of activating transcription factor 4 (ATF4). During wild-type HCMV infection, increases in Xbp-1 splicing, eIF2α phosphorylation, and accumulation of ATF4 accompany UL148 expression. UL148-null infections, however, show reduced levels of these UPR indicators and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. Small interfering RNA (siRNA) depletion of PERK dampened the extent of eIF2α phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2α kinases. A virus with UL148 disrupted showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV-infected cell.IMPORTANCE The unfolded protein response (UPR) is an ancient cellular response to ER stress that is of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shutoff, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER-resident glycoprotein UL148 contributes substantially to the UPR during infection and, moreover, is sufficient to activate the UPR in noninfected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell-mediated immunity.

Siddiquey MNA ，Zhang H ，Nguyen CC ，Domma AJ ，Kamil JP ... -  《-》

Human cytomegalovirus infection activates and regulates the unfolded protein response.

Isler JA ，Skalet AH ，Alwine JC 《-》

The Human Cytomegalovirus Nonstructural Glycoprotein UL148 Reorganizes the Endoplasmic Reticulum.

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident glycoprotein, UL148, which activates the unfolded protein response (UPR) but is fully dispensable for viral replication in cultured cells. Hence, its previously ascribed roles in immune evasion and modulation of viral cell tropism are hypothesized to cause ER stress. Here, we show that UL148 is necessary and sufficient to drive the formation of prominent ER-derived structures that on average occupy 5% of the infected cell cytoplasm. The structures are sites where UL148 coalesces with cellular proteins involved in ER quality control, such as HRD1 and EDEM1. Electron microscopy revealed that cells infected with wild-type but not UL148-null HCMV show prominent accumulations of densely packed ruffled ER membranes which connect to distended cisternae of smooth and partially rough ER. During ectopic expression of UL148-green fluorescent protein (GFP) fusion protein, punctate signals traffic to accumulate at conspicuous structures. The structures exhibit poor recovery of fluorescence after photobleaching, which suggests that their contents are poorly mobile and do not efficiently exchange with the rest of the ER. Small-molecule blockade of the integrated stress response (ISR) prevents the formation of puncta, leading to a uniform reticular fluorescent signal. Accordingly, ISR inhibition during HCMV infection abolishes the coalescence of UL148 and HRD1 into discrete structures, which argues that UL148 requires the ISR to cause ER reorganization. Given that UL148 stabilizes immature forms of a receptor binding subunit for a viral envelope glycoprotein complex important for HCMV infectivity, our results imply that stress-dependent ER remodeling contributes to viral cell tropism.IMPORTANCE Perturbations to endoplasmic reticulum (ER) morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that a human cytomegalovirus (HCMV) gene product, UL148, profoundly reorganizes the ER during infection and is sufficient to do so when expressed on its own. Our results reveal that UL148-dependent reorganization of the ER is a prominent feature of HCMV-infected cells. Moreover, we find that this example of virally induced organelle remodeling requires the integrated stress response (ISR), a stress adaptation pathway that contributes to a number of disease states. Since ER reorganization accompanies roles of UL148 in modulation of HCMV cell tropism and in evasion of antiviral immune responses, our results may have implications for understanding the mechanisms involved. Furthermore, our findings provide a basis to utilize UL148 as a tool to investigate organelle responses to stress and to identify novel drugs targeting the ISR.

Zhang H ，Read C ，Nguyen CC ，Siddiquey MNA ，Shang C ，Hall CM ，von Einem J ，Kamil JP ... -  《mBio》

Endoplasmic Reticulum (ER) Reorganization and Intracellular Retention of CD58 Are Functionally Independent Properties of the Human Cytomegalovirus ER-Resident Glycoprotein UL148.

The human cytomegalovirus (HCMV) endoplasmic reticulum (ER)-resident glycoprotein UL148 is posited to play roles in immune evasion and regulation of viral cell tropism. UL148 prevents cell surface presentation of the immune cell costimulatory ligand CD58 while promoting maturation and virion incorporation of glycoprotein O, a receptor binding subunit for an envelope glycoprotein complex involved in entry. Meanwhile, UL148 activates the unfolded protein response (UPR) and causes large-scale reorganization of the ER. In order to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated six charged cluster-to-alanine (CCTA) mutants within the UL148 ectodomain and compared them to wild-type UL148, both in the context of infection studies using recombinant viruses and in ectopic expression experiments, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79 to 83 (CC3) and 133 to 136 (CC4), retained the potential to impede CD58 surface presentation. Of the six mutants, only CC3 retained the capacity to reorganize the ER, but it showed a partial phenotype. Wild-type UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which implies a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses surface presentation of CD58 but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate various degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that the capacity to cause ER reorganization is unique to HCMV UL148.IMPORTANCE In myriad examples, viral gene products cause striking effects on cells, such as activation of stress responses. It can be challenging to decipher how such effects contribute to the biological roles of the proteins. The HCMV glycoprotein UL148 retains CD58 within the ER, thereby preventing it from reaching the cell surface, where it functions to stimulate cell-mediated antiviral responses. Intriguingly, UL148 also triggers the formation of large, ER-derived membranous structures and activates the UPR, a set of signaling pathways involved in adaptation to ER stress. We demonstrate that the potential of UL148 to reorganize the ER and to retain CD58 are separable by mutagenesis and, possibly, by evolution, since chimpanzee cytomegalovirus UL148 retains CD58 but does not remodel the ER. Our findings imply that ER reorganization contributes to other roles of UL148, such as modulation of alternative viral glycoprotein complexes that govern the virus' ability to infect different cell types.

Nguyen CC ，Domma AJ ，Zhang H ，Kamil JP ... -  《-》

The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress.

Teske BF ，Wek SA ，Bunpo P ，Cundiff JK ，McClintick JN ，Anthony TG ，Wek RC ... -  《-》