scholarly journals The Human Cytomegalovirus Nonstructural Glycoprotein UL148 Reorganizes the Endoplasmic Reticulum

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Hongbo Zhang ◽  
Clarissa Read ◽  
Christopher C. Nguyen ◽  
Mohammed N. A. Siddiquey ◽  
Chaowei Shang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Viral Envelope ◽  
Cell Tropism ◽  
Integrated Stress Response ◽  
Er Quality Control ◽  
Stress Dependent

ABSTRACT 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.

scholarly journals A cytomegalovirus immunevasin triggers integrated stress response-dependent reorganization of the endoplasmic reticulum

2019 ◽  
Author(s):  
Hongbo Zhang ◽  
Clarissa Read ◽  
Christopher C. Nguyen ◽  
Mohammed N.A. Siddiquey ◽  
Chaowei Shang ◽  
...  
Keyword(s):  
Stress Response ◽  
Immune Cell ◽  
Cultured Cells ◽  
Genetic Disorders ◽  
Ectopic Expression ◽  
Viral Envelope ◽  
Cell Tropism ◽  
Integrated Stress Response ◽  
Er Quality Control ◽  
Stress Dependent

ABSTRACTHuman cytomegalovirus (HCMV) encodes an 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 large ER-derived structures that occupy up to 7% of the infected cell cytosol. The structures are found to be sites where UL148 coalesces with cellular proteins involved in ER quality control, such as Hrd1 and EDEM1. Transmission electron microscopy analyses reveal the structures to be comprised of tortuous, densely packed and apparently collapsed ER membranes that connect to distended cisternae. During induced ectopic expression of UL148-GFP fusion protein, punctate signals traffic to accumulate at prominent structures that exhibit poor recovery of fluorescence after photobleaching. 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.IMPORTANCEPerturbations to ER morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that an 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 the roles of UL148 in HCMV cell tropism and intracellular retention of the immune cell co-stimulatory ligand CD58, 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.

scholarly journals Human Cytomegalovirus Tropism Modulator UL148 Interacts with SEL1L, a Cellular Factor That Governs Endoplasmic Reticulum-Associated Degradation of the Viral Envelope Glycoprotein gO

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Christopher C. Nguyen ◽  
Mohammed N. A. Siddiquey ◽  
Hongbo Zhang ◽  
Gang Li ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Host Cells ◽  
Cell Tropism ◽  
Viral Glycoprotein ◽  
Viral Envelope Glycoprotein ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Viral Glycoproteins

ABSTRACTUL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into the mechanism, we used mass spectrometry to identify proteins that coimmunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected withUL148-null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, small interfering RNA (siRNA) silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERAD substrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism.IMPORTANCEViral glycoproteins in large part determine the cell types that an enveloped virus can infect and hence play crucial roles in transmission and pathogenesis. The glycoprotein H/L heterodimer (gH/gL) is part of the conserved membrane fusion machinery that all herpesviruses use to enter cells. In human cytomegalovirus (HCMV), gH/gL participates in alternative complexes in virions, one of which is a trimer of gH/gL with glycoprotein O (gO). Here, we show that gO is constitutively degraded during infection by the endoplasmic reticulum-associated degradation (ERAD) pathway and that UL148, a viral factor that regulates HCMV cell tropism, interacts with the ERAD machinery and slows gO decay. Since gO is required for cell-free virus to enter new host cells but dispensable for cell-associated spread that resists antibody neutralization, our findings imply that the posttranslational instability of a viral glycoprotein provides a basis for viral mechanisms to modulate tropism and spread.

scholarly journals Viral Regulation of Cell Tropism in Human Cytomegalovirus

2015 ◽  
Vol 90 (2) ◽  
pp. 626-629 ◽  
Author(s):  
Gang Li ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Cell Types ◽  
Viral Envelope ◽  
Cell Entry ◽  
Cell Tropism ◽  
Enveloped Viruses ◽  
Viral Envelope Glycoprotein ◽  
Viral Glycoproteins

The viral glycoproteins that decorate enveloped viruses play crucial roles in cell entry and in large part dictate the spectrum of cell types that a virus can infect. The identification in human cytomegalovirus (HCMV) of a viral endoplasmic reticulum (ER)-resident glycoprotein that regulates the composition of alternative viral envelope glycoprotein complexes raises the intriguing possibility that certain viruses might actively regulate the tropism of progeny virions to improve their fitness or to navigate through the host.

scholarly journals Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Cheng Wang ◽  
Zhijia Tan ◽  
Ben Niu ◽  
Kwok Yeung Tsang ◽  
Andrew Tai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Cell Fate ◽  
Therapeutic Strategy ◽  
Chondrocyte Differentiation ◽  
Integrated Stress Response ◽  
Stress Response Pathway ◽  
Skeletal Disorders ◽  
The Impact ◽  
Insight Into

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

scholarly journals Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Danielle M Garshott ◽  
Elayanambi Sundaramoorthy ◽  
Marilyn Leonard ◽  
Eric J Bennett
Keyword(s):  
Quality Control ◽  
Stress Response ◽  
Ribosomal Proteins ◽  
Functional Role ◽  
Stress Recovery ◽  
Integrated Stress Response ◽  
Hierarchical Relationship ◽  
Stress Dependent

Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role and fate of ubiquitylated proteins remain unclear. We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation, respectively, suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes. We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation. Critically, cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway.

scholarly journals Endoplasmic reticulum chaperones participate in human cytomegalovirus US2-mediated degradation of class I major histocompatibility complex molecules

2008 ◽  
Vol 89 (5) ◽  
pp. 1122-1130 ◽  
Author(s):  
Kristina Oresic ◽  
Domenico Tortorella
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Major Histocompatibility Complex ◽  
Human Cytomegalovirus ◽  
Class I ◽  
Cell Surface Expression ◽  
Major Histocompatibility ◽  
Er Quality Control ◽  
Heavy Chains ◽  
Histocompatibility Complex

Inhibition of cell-surface expression of major histocompatibility complex class I molecules by human cytomegalovirus (HCMV, a β-herpesvirus) promotes escape from recognition by CD8+ cytotoxic T cells. The HCMV US2 and US11 gene products induce class I downregulation during the early phase of HCMV infection by facilitating the degradation of class I heavy chains. The HCMV proteins promote the transport of the class I heavy chains across the endoplasmic reticulum (ER) membrane into the cytosol by a process referred to as ‘dislocation’, which is then followed by proteasome degradation. This process has striking similarities to the degradation of misfolded ER proteins mediated by ER quality control. Even though the major steps of the dislocation reaction have been characterized, the cellular proteins, specifically the ER chaperones involved in targeting class I for dislocation, have not been fully delineated. To elucidate the chaperones involved in HCMV-mediated class I dislocation, we utilized a chimeric class I heavy chain with an affinity tag at its carboxy terminus. Interestingly, US2 but not US11 continued to target the class I chimera for destruction, suggesting a structural limitation for US11-mediated degradation. Association studies in US2 cells and in cells that express a US2 mutant, US2–186HA, revealed that class I specifically interacts with calnexin, BiP and calreticulin. These findings demonstrate that US2-mediated class I destruction utilizes specific chaperones to facilitate class I dislocation. The data suggest a more general model in which the chaperones that mediate protein folding may also function during ER quality control to eliminate aberrant ER proteins.

scholarly journals Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 848 ◽  
Author(s):  
Pingan Chang ◽  
Ling He ◽  
Yu Wang ◽  
Christoph Heier ◽  
Yijun Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Terminal Region ◽  
Neuropathy Target Esterase ◽  
Loss Of Function ◽  
Dynamic Interactions ◽  
Functional Regions ◽  
R Region

Neuropathy target esterase (NTE) is an endoplasmic reticulum (ER)-localized phospholipase that deacylates phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Loss-of-function mutations in the human NTE gene have been associated with a spectrum of neurodegenerative disorders such as hereditary spastic paraplegia, ataxia and chorioretinal dystrophy. Despite this, little is known about structure–function relationships between NTE protein domains, enzymatic activity and the interaction with cellular organelles. In the current study we show that the C-terminal region of NTE forms a catalytically active domain that exhibits high affinity for lipid droplets (LDs), cellular storage organelles for triacylglycerol (TAG), which have been recently implicated in the progression of neurodegenerative diseases. Ectopic expression of the C domain in cultured cells decreases cellular PC, elevates TAG and induces LD clustering. LD interactions of NTE are inhibited by default by a non-enzymatic regulatory (R) region with three putative nucleotide monophosphate binding sites. Together with a N-terminal TMD the R region promotes proper distribution of the catalytic C-terminal region to the ER network. Taken together, our data indicate that NTE may exhibit dynamic interactions with the ER and LDs depending on the interplay of its functional regions. Mutations that disrupt this interplay may contribute to NTE-associated disorders by affecting NTE positioning.

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

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Christopher C. Nguyen ◽  
Anthony J. Domma ◽  
Hongbo Zhang ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Human Cytomegalovirus ◽  
Stress Responses ◽  
Immune Cell ◽  
Functional Divergence ◽  
Ectopic Expression ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Glycoprotein

ABSTRACT 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.

scholarly journals eIF2B-capturing viral protein NSs suppresses the integrated stress response

2021 ◽  
Author(s):  
Kazuhiro Kashiwagi ◽  
Yuichi Shichino ◽  
Tatsuya Osaki ◽  
Ayako Sakamoto ◽  
Madoka Nishimoto ◽  
...  
Keyword(s):  
Stress Response ◽  
Cultured Cells ◽  
Nonstructural Protein ◽  
Ribosome Profiling ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Neuroprotective Effects ◽  
Α Subunit ◽  
A Genome

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotides on unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. Our cryo-electron microscopy (cryo-EM) analysis revealed that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B exhibits normal nucleotide exchange activity even in the presence of eIF2(αP). A genome-wide ribosome profiling analysis clarified that SFSV NSs in human cultured cells attenuates the ISR. Furthermore, SFSV NSs exhibited neuroprotective effects against the ISR-inducing stress. Since the ISR inhibition is beneficial in various neurological disease models, SFSV NSs is promising as a therapeutic ISR inhibitor.

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