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

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Mohammed N. A. Siddiquey ◽  
Hongbo Zhang ◽  
Christopher C. Nguyen ◽  
Anthony J. Domma ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Human Cytomegalovirus ◽  
Mrna Translation ◽  
Wild Type ◽  
Α Subunit ◽  
Unfolded Protein ◽  
Eif2α Phosphorylation ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTEukaryotic 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 ofXbp-1mRNA, 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 inXbp-1splicing, 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 withUL148disrupted 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.IMPORTANCEThe 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.

scholarly journals The human cytomegalovirus ER resident glycoprotein UL148 activates the unfolded protein response

2018 ◽  
Author(s):  
Mohammed N.A. Siddiquey ◽  
Hongbo Zhang ◽  
Christopher C. Nguyen ◽  
Anthony J. Domma ◽  
Jeremy P. Kamil
Keyword(s):  
Unfolded Protein Response ◽  
Human Cytomegalovirus ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Mrna Translation ◽  
Cell Mediated Immunity ◽  
Wild Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTEukaryotic 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 markedly decreased when UL148 expression is induced in uninfected cells. Further, we find evidence suggesting that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol requiring enzyme-1 (IRE1), as indicated by splicing ofXbp1mRNA, and the PKR-like ER kinase (PERK), as indicated by phosphorylation of eIF2αand accumulation of ATF4 protein. During wild-type HCMV infection,Xbp-1splicing, eIF2αphosphorylation and ATF4 accumulation neatly accompanied the onset of UL148 expression. However, the appearance of these UPR indicators was either markedly delayed or absent duringUL148-null infections. siRNA depletion of PERK dampened the extent of eIF2αphosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2αkinases. A virus disrupted forUL148showed statistically 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.IMPORTANCEThe unfolded protein response (UPR) is an ancient cellular response to ER stress of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shut-off, 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 at least 200 distinct 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 non-infected 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.

scholarly journals Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response

2005 ◽  
Vol 79 (11) ◽  
pp. 6890-6899 ◽  
Author(s):  
Jennifer A. Isler ◽  
Alison H. Skalet ◽  
James C. Alwine
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Endoplasmic Reticulum Stress ◽  
Viral Infection ◽  
Unfolded Protein Response ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.

scholarly journals Human Cytomegalovirus Protein pUL38 Induces ATF4 Expression, Inhibits Persistent JNK Phosphorylation, and Suppresses Endoplasmic Reticulum Stress-Induced Cell Death

2009 ◽  
Vol 83 (8) ◽  
pp. 3463-3474 ◽  
Author(s):  
Baoqin Xuan ◽  
Zhikang Qian ◽  
Emi Torigoi ◽  
Dong Yu
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Human Cytomegalovirus ◽  
Initiation Factor ◽  
Α Subunit ◽  
Unfolded Protein ◽  
Initiation Factor 2 ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT The endoplasmic reticulum (ER) is a key organelle involved in sensing and responding to stressful conditions, including those resulting from infection of viruses, such as human cytomegalovirus (HCMV). Three signaling pathways collectively termed the unfolded protein response (UPR) are activated to resolve ER stress, but they will also lead to cell death if the stress cannot be alleviated. HCMV is able to modulate the UPR to promote its infection. The specific viral factors involved in such HCMV-mediated modulation, however, were unknown. We previously showed that HCMV protein pUL38 was required to maintain the viability of infected cells, and it blocked cell death induced by thapsigargin. Here, we report that pUL38 is an HCMV-encoded regulator to modulate the UPR. In infection, pUL38 allowed HCMV to upregulate phosphorylation of PKR-like ER kinase (PERK) and the α subunit of eukaryotic initiation factor 2 (eIF-2α), as well as induce robust accumulation of activating transcriptional factor 4 (ATF4), key components of the PERK pathway. pUL38 also allowed the virus to suppress persistent phosphorylation of c-Jun N-terminal kinase (JNK), which was induced by the inositol-requiring enzyme 1 pathway. In isolation, pUL38 overexpression elevated eIF-2α phosphorylation, induced ATF4 accumulation, limited JNK phosphorylation, and suppressed cell death induced by both thapsigargin and tunicamycin, two drugs that induce ER stress by different mechanisms. Importantly, ATF4 overexpression and JNK inhibition significantly reduced cell death in pUL38-deficient virus infection. Thus, pUL38 targets ATF4 expression and JNK activation, and this activity appears to be critical for protecting cells from ER stress induced by HCMV infection.

Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic

2001 ◽  
Vol 114 (20) ◽  
pp. 3685-3694
Author(s):  
Thomas K. Graves ◽  
Shilpa Patel ◽  
Priscilla S. Dannies ◽  
Patricia M. Hinkle
Keyword(s):  
Growth Hormone ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Unfolded Protein Response ◽  
Wild Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Trh Receptor ◽  
In Cells

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Δ32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Δ32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Δ32-71 growth hormone were dually stained for growth hormone and the Golgi markers β-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but β-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Δ32-71 growth hormone. Examination of α-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Δ32-71 growth hormone. To determine whether Δ32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Δ32-71 growth hormone. Cells expressing Δ32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Δ32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Δ32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

scholarly journals Stable Ribosome Binding to the Endoplasmic Reticulum Enables Compartment-specific Regulation of mRNA Translation

2005 ◽  
Vol 16 (12) ◽  
pp. 5819-5831 ◽  
Author(s):  
Samuel B. Stephens ◽  
Rebecca D. Dodd ◽  
Joseph W. Brewer ◽  
Patrick J. Lager ◽  
Jack D. Keene ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Unfolded Protein Response ◽  
Stress Proteins ◽  
Signal Sequence ◽  
Mrna Translation ◽  
Physiological Basis ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

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