The Autophagy-Initiating Protein Kinase ULK1 Phosphorylates Human Cytomegalovirus Tegument Protein pp28 and Regulates Efficient Virus Release.
TL;DR: The study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit.
Abstract: Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.
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TL;DR: Considering the COVID-19 pandemic, understanding the regulation of AMPK signaling following infection can shed light on the development of more effective therapeutic strategies against viral infectious diseases.
Abstract: Viral pathogens often exploit host cell regulatory and signaling pathways to ensure an optimal environment for growth and survival. Several studies have suggested that 5′-adenosine monophosphate-activated protein kinase (AMPK), an intracellular serine/threonine kinase, plays a significant role in the modulation of infection. Traditionally, AMPK is a key energy regulator of cell growth and proliferation, host autophagy, stress responses, metabolic reprogramming, mitochondrial homeostasis, fatty acid β-oxidation and host immune function. In this review, we highlight the modulation of host AMPK by various viruses under physiological conditions. These intracellular pathogens trigger metabolic changes altering AMPK signaling activity that then facilitates or inhibits viral replication. Considering the COVID-19 pandemic, understanding the regulation of AMPK signaling following infection can shed light on the development of more effective therapeutic strategies against viral infectious diseases.
23 citations
Cites background from "The Autophagy-Initiating Protein Ki..."
...Viruses use AMPK to manipulate autophagy [7,8], fatty acid and lipid metabolism [5,9], glucose metabolism [10,11] and many other cellular processes....
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...Indeed, regulated autophagy plays an important role in HCMV life cycle and autophagy-initiating protein kinase ULK1 has been found to phosphorylate the HCMV tegument protein pp28 and to regulate virion release [8]....
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TL;DR: In this article, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood, and it is shown that PERK is an essential host factor that supports HCM virus replication and promotes lipidome changes caused by HCMv infection.
Abstract: Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.
10 citations
TL;DR: The role of H CMV in gliomagenesis, disease prognosis, and recent breakthroughs in harnessing HCMV-induced immunogenicity in the GB tumor microenvironment to develop effective CMV-specific ACT are discussed.
Abstract: A high percentage of malignant gliomas are infected by human cytomegalovirus (HCMV), and the endogenous expression of HCMV genes and their products are found in these tumors. HCMV antigen expression and its implications in gliomagenesis have emerged as a promising target for adoptive cellular immunotherapy (ACT) strategies in glioblastoma multiforme (GB) patients. Since antigen-specific T cells in the tumor microenvironments lack efficient anti-tumor immune response due to the immunosuppressive nature of glioblastoma, CMV-specific ACT relies on in vitro expansion of CMV-specific CD8+ T cells employing immunodominant HCMV antigens. Given the fact that several hurdles remain to be conquered, recent clinical trials have outlined the feasibility of CMV-specific ACT prior to tumor recurrence with minimal adverse effects and a substantial improvement in median overall survival and progression-free survival. This review discusses the role of HCMV in gliomagenesis, disease prognosis, and recent breakthroughs in harnessing HCMV-induced immunogenicity in the GB tumor microenvironment to develop effective CMV-specific ACT.
9 citations
TL;DR: In this paper , a review of the interaction between autophagy and viruses is presented, which explains how autophage serves multiple roles in viral infection, with either proviral or antiviral functions.
Abstract: Autophagy is an evolutionarily conserved catabolic cellular process that exerts antiviral functions during a viral invasion. However, co-evolution and co-adaptation between viruses and autophagy have armed viruses with multiple strategies to subvert the autophagic machinery and counteract cellular antiviral responses. Specifically, the host cell quickly initiates the autophagy to degrade virus particles or virus components upon a viral infection, while cooperating with anti-viral interferon response to inhibit the virus replication. Degraded virus-derived antigens can be presented to T lymphocytes to orchestrate the adaptive immune response. Nevertheless, some viruses have evolved the ability to inhibit autophagy in order to evade degradation and immune responses. Others induce autophagy, but then hijack autophagosomes as a replication site, or hijack the secretion autophagy pathway to promote maturation and egress of virus particles, thereby increasing replication and transmission efficiency. Interestingly, different viruses have unique strategies to counteract different types of selective autophagy, such as exploiting autophagy to regulate organelle degradation, metabolic processes, and immune responses. In short, this review focuses on the interaction between autophagy and viruses, explaining how autophagy serves multiple roles in viral infection, with either proviral or antiviral functions.
5 citations
TL;DR: The role of stress in HCMV reprogramming of lipid metabolism is poorly understood as discussed by the authors, and the role of ER stress mediator, namely PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMVs infection.
Abstract: Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions. IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.
5 citations
References
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TL;DR: A molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1, is demonstrated and a signalling mechanism for UlK1 regulation and autophagic induction in response to nutrient signalling is revealed.
Abstract: Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
5,314 citations
"The Autophagy-Initiating Protein Ki..." refers background in this paper
...S758, AMPK-dependent activating as well as mTOR-dependent inactivating phosphorylation sites of ULK1 were affected equally (29)....
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TL;DR: The molecular mechanism of autophagosome formation is described with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagous membrane membrane.
Abstract: Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.
2,522 citations
"The Autophagy-Initiating Protein Ki..." refers background in this paper
...the only protein kinase of the core autophagy machinery (19)....
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TL;DR: Reconstitution of ULK1-deficient cells with a mutant ULK2 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation.
Abstract: Adenosine monophosphate–activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.
2,137 citations
TL;DR: It is identified that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2, which demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
Abstract: Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
1,801 citations
"The Autophagy-Initiating Protein Ki..." refers background or methods or result in this paper
...The expression plasmid encoding HA-ULK1 was generated by amplification of the ULK1 coding sequence from pRK5_myc-ULK1 (a gift from Do-Hyung Kim; Addgene plasmid 31961) (22) utilizing 59-GCATGAATTCATGTACCCATACGATGTTCCAGATTACGCTGAGC CCGGCCGCGGCGGCACAG-39 (containing the HA sequence) and 59-CGTATCTAGATTAGGCACAGATGCC AGTCAGC-39 as primers and subsequent ligation into pcDNA3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) with EcoRI and XbaI....
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...pRK5_myc-ATG13 was a gift from Do-Hyung Kim (Addgene plasmid 31965) (22)....
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...This is consistent with previous reports indicating that ATG13 as part of the ULK1 protein complex affects the stability of ULK1 (22, 25)....
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TL;DR: Recent advances in identifying and understanding the core molecular machinery and signaling pathways that are involved in mammalian autophagy are highlighted.
1,782 citations