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1
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Kalamvoki M. HSV-1 virions and related particles: biogenesis and implications in the infection. J Virol 2025; 99:e0107624. [PMID: 39898651 PMCID: PMC11915793 DOI: 10.1128/jvi.01076-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2025] Open
Abstract
Virion formation and egress are sophisticated processes that rely on the spatial and temporal organization of host cell membranes and the manipulation of host machineries involved in protein sorting, membrane bending, fusion, and fission. These processes result in the formation of infectious virions, defective particles, and various vesicle-like structures. In herpes simplex virus 1 (HSV-1) infections, virions and capsid-less particles, known as light (L)-particles, are formed. HSV-1 infection also stimulates the release of particles that resemble extracellular vesicles (EVs). In productively infected cells, most EVs are generated through the CD63 tetraspanin biogenesis pathway and lack viral components. A smaller subset of EVs, generated through the endosomal sorting complexes required for transport (ESCRT) pathway, contains both viral and host factors. Viral mechanisms tightly regulate EV biogenesis, including the inhibition of autophagy-a process critical for increased production of CD63+ EVs during HSV-1 infection. Mutant viruses that fail to suppress autophagy instead promote microvesicle production from the plasma membrane. Additionally, the viral protein ICP0 (Infected Cell Protein 0) enhances EV biogenesis during HSV-1 infection. The different types of particles can be separated by density gradients due to their distinct biophysical properties. L-particles and ESCRT+ EVs display a pro-viral role, supporting viral replication, whereas CD63+ EVs exhibit antiviral effects. Overall, these studies highlight that HSV-1 infection yields numerous and diverse particles, with their type and composition shaped by the ability of the virus to evade host responses. These particles likely shape the infectious microenvironment and determine disease outcomes.
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Affiliation(s)
- Maria Kalamvoki
- Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
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2
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Wang D, Chen D, Xu S, Wei F, Zhao H. Comparative proteomic analysis of PK-15 cells infected with wild-type strain and its EP0 gene-deleted mutant strain of pseudorabies virus. J Vet Sci 2024; 25:e54. [PMID: 39083206 PMCID: PMC11291433 DOI: 10.4142/jvs.24069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Revised: 05/27/2024] [Accepted: 06/06/2024] [Indexed: 08/02/2024] Open
Abstract
IMPORTANCE As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear. OBJECTIVE This study examined the function of EP0 to provide a direction for its in-depth analysis. METHODS In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells. RESULTS This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization. CONCLUSIONS AND RELEVANCE These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections.
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Affiliation(s)
- Di Wang
- School of Agroforestry and Medicine, The Open University of China, Beijing 100039, China
| | - Dongjie Chen
- Institute of Animal Inspection and Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, China.
| | - Shengkui Xu
- College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China
| | - Fang Wei
- Institute of Animal Inspection and Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, China
| | - Hongyuan Zhao
- School of Modern Agriculture & Biotechnology, Ankang University, Ankang 725000, China.
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3
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Pietilä MK, Bachmann JJ, Ravantti J, Pelkmans L, Fraefel C. Cellular state landscape and herpes simplex virus type 1 infection progression are connected. Nat Commun 2023; 14:4515. [PMID: 37500668 PMCID: PMC10374626 DOI: 10.1038/s41467-023-40148-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2022] [Accepted: 07/14/2023] [Indexed: 07/29/2023] Open
Abstract
Prediction, prevention and treatment of virus infections require understanding of cell-to-cell variability that leads to heterogenous disease outcomes, but the source of this heterogeneity has yet to be clarified. To study the multimodal response of single human cells to herpes simplex virus type 1 (HSV-1) infection, we mapped high-dimensional viral and cellular state spaces throughout the infection using multiplexed imaging and quantitative single-cell measurements of viral and cellular mRNAs and proteins. Here we show that the high-dimensional cellular state scape can predict heterogenous infections, and cells move through the cellular state landscape according to infection progression. Spatial information reveals that infection changes the cellular state of both infected cells and of their neighbors. The multiplexed imaging of HSV-1-induced cellular modifications links infection progression to changes in signaling responses, transcriptional activity, and processing bodies. Our data show that multiplexed quantification of responses at the single-cell level, across thousands of cells helps predict infections and identify new targets for antivirals.
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Affiliation(s)
- Maija K Pietilä
- Institute of Virology, University of Zurich, Zurich, Switzerland.
| | - Jana J Bachmann
- Institute of Virology, University of Zurich, Zurich, Switzerland
| | - Janne Ravantti
- Molecular and Integrative Biosciences Research Programme, University of Helsinki, Helsinki, Finland
| | - Lucas Pelkmans
- Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
| | - Cornel Fraefel
- Institute of Virology, University of Zurich, Zurich, Switzerland.
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4
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Harrell TL, Davido DJ, Bertke AS. Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 0 (ICP0) Targets of Ubiquitination during Productive Infection of Primary Adult Sensory Neurons. Int J Mol Sci 2023; 24:2931. [PMID: 36769256 PMCID: PMC9917815 DOI: 10.3390/ijms24032931] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Revised: 01/18/2023] [Accepted: 01/26/2023] [Indexed: 02/05/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons.
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Affiliation(s)
- Telvin L. Harrell
- Biomedical and Veterinary Science, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
| | - David J. Davido
- Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
| | - Andrea S. Bertke
- Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
- Center for Emerging Zoonotic and Arthropod-Borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
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5
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Dong H, Wu W, Li J, Ma Y, Deng X, Guo D, Xu P. PML Body Component Sp100A Is a Cytosolic Responder to IFN and Activator of Antiviral ISGs. mBio 2022; 13:e0204422. [PMID: 36383022 PMCID: PMC9765618 DOI: 10.1128/mbio.02044-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2022] [Accepted: 10/28/2022] [Indexed: 11/18/2022] Open
Abstract
Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive. Herein, we report that a major constitutive component of the PML body, Sp100A, is ubiquitously located in the cytosol of various cell types and is an immediate responder to multiple extracellular stimuli, including virus infection, IFN, epidermal growth factor (EGF), glial cell-derived nerve factor (GDNF), etc., signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. IFN-β induces phosphorylation of Sp100A on Ser188, which fortifies the binding of Sp100A to pyruvate kinase 2 (PKM2) and facilitates its nuclear importation through the extracellular signal-regulated kinase 1/2 (ERK1/2)-PKM2-PIN1-importin axes. Blocking PI3K pathway signaling or interference with the ERK1/2-PKM2-PIN1-importin axes independently hampers nuclear translocation of Sp100A in response to IFN, reflecting a dual-regulation mechanism governing this event. In the nucleus, Sp100A is enriched in the promoter regions of essential antiviral interferon-stimulated genes (ISGs), such as those coding for IFI16, OAS2, and RIG-I, and activates their transcription. Importantly, nuclear importation of Sp100A, but not accumulation of a mutant Sp100A that failed to respond to IFN, during infection potently enhanced transcription of these antiviral ISGs and restricted virus propagation. These findings depict a novel IFN response mechanism by PML bodies in the cytosol and shed light on the complex sensing-regulatory network of PML bodies. IMPORTANCE PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection. It shows that PML body constitutive component Sp100A was phosphorylated on Ser188 by IFN signaling through the PI3K pathway in the cytosol, cotranslocated into the nucleus with PKM2, enriched on the promoter regions of essential antiviral ISGs such as those coding for IFI16, RIG-I, OAS2, etc., and mediating their transcriptional activation.
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Affiliation(s)
- Hongchang Dong
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
| | - Wencheng Wu
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
| | - Jingjing Li
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
| | - Yilei Ma
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
| | - Xiaomei Deng
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
| | - Deyin Guo
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
- Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University, Guangzhou, People’s Republic of China
| | - Pei Xu
- The Centre for Infection and Immunity Studies, School of Medicine, Sun Yat-sen University, Shenzhen, People’s Republic of China
- Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University, Guangzhou, People’s Republic of China
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6
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Ma XH, Yao YX, Wang XZ, Zhou YP, Huang SN, Li D, Mei MJ, Wu JP, Pan YT, Cheng S, Jiang X, Sun JY, Zeng WB, Gong S, Cheng H, Luo MH, Yang B. MORC3 restricts human cytomegalovirus infection by suppressing the major immediate-early promoter activity. J Med Virol 2022; 94:5492-5506. [PMID: 35879101 DOI: 10.1002/jmv.28025] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 07/14/2022] [Accepted: 07/21/2022] [Indexed: 12/15/2022]
Abstract
During the long coevolution of human cytomegalovirus (HCMV) and humans, the host has formed a defense system of multiple layers to eradicate the invader, and the virus has developed various strategies to evade host surveillance programs. The intrinsic immunity primarily orchestrated by promyelocytic leukemia (PML) nuclear bodies (PML-NBs) represents the first line of defense against HCMV infection. Here, we demonstrate that microrchidia family CW-type zinc finger 3 (MORC3), a PML-NBs component, is a restriction factor targeting HCMV infection. We show that depletion of MORC3 through knockdown by RNA interference or knockout by CRISPR-Cas9 augmented immediate-early protein 1 (IE1) gene expression and subsequent viral replication, and overexpressing MORC3 inhibited HCMV replication by suppressing IE1 gene expression. To relief the restriction, HCMV induces transient reduction of MORC3 protein level via the ubiquitin-proteasome pathway during the immediate-early to early stage. However, MORC3 transcription is upregulated, and the protein level recovers in the late stages. Further analyses with temporal-controlled MORC3 expression and the major immediate-early promoter (MIEP)-based reporters show that MORC3 suppresses MIEP activity and consequent IE1 expression with the assistance of PML. Taken together, our data reveal that HCMV enforces temporary loss of MORC3 to evade its repression against the initiation of immediate-early gene expression.
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Affiliation(s)
- Xue-Hui Ma
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China
| | - Yong-Xuan Yao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Xian-Zhang Wang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Yue-Peng Zhou
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Sheng-Nan Huang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Dong Li
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Meng-Jie Mei
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Jing-Peng Wu
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Yu-Ting Pan
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Shuang Cheng
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China
| | - Xuan Jiang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Jin-Yan Sun
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China
| | - Wen-Bo Zeng
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China
| | - Sitang Gong
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Han Cheng
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Min-Hua Luo
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,University of Chinese Academy of Sciences, Beijing, China.,Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Bo Yang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, China.,State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
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7
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Ma Y, Li J, Dong H, Yang Z, Zhou L, Xu P. PML Body Component Sp100A Restricts Wild-Type Herpes Simplex Virus 1 Infection. J Virol 2022; 96:e0027922. [PMID: 35353002 PMCID: PMC9044927 DOI: 10.1128/jvi.00279-22] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2022] [Accepted: 03/07/2022] [Indexed: 12/13/2022] Open
Abstract
Sp100 (speckled protein 100 kDa) is a constituent component of nuclear structure PML (promyelocytic leukemia) bodies, playing important roles in mediating intrinsic and innate immunity. The Sp100 gene encodes four isoforms with distinct roles in the transcriptional regulation of both cellular and viral genes. Since Sp100 is a primary intranuclear target of infected-cell protein 0 (ICP0), an immediate early E3 ligase encoded by herpes simplex virus 1 (HSV-1), previous investigations attempting to analyze the functions of individual Sp100 variants during HSV-1 infection mostly avoided using a wild-type virus. Therefore, the role of Sp100 under natural infection by HSV-1 remains to be clarified. Here, we reappraised the antiviral capacity of four Sp100 isoforms during infection by a nonmutated HSV-1, examined the molecular behavior of the Sp100 protein in detail, and revealed the following intriguing observations. First, Sp100 isoform A (Sp100A) inhibited wild-type HSV-1 propagation in HEp-2, Sp100-/-, and PML-/- cells. Second, endogenous Sp100 is located in both the nucleus and the cytoplasm. During HSV-1 infection, the nuclear Sp100 level decreased drastically upon the detection of ICP0 in the same subcellular compartment, but cytosolic Sp100 remained stable. Third, transfected Sp100A showed subcellular localizations similar to those of endogenous Sp100 and matched the protein size of endogenous cytosolic Sp100. Fourth, HSV-1 infection induced increased secretion of endogenous Sp100 and ectopically expressed Sp100A, which copurified with extracellular vesicles (EVs) but not infectious virions. Fifth, the Sp100A level in secreting cells positively correlated with its level in EVs, and EV-associated Sp100A restricted HSV-1 in recipient cells. IMPORTANCE Previous studies show that the PML body component Sp100 protein is immediately targeted by ICP0 of HSV-1 in the nucleus during productive infection. Therefore, extensive studies investigating the interplay of Sp100 isoforms with HSV-1 were conducted using a mutant virus lacking ICP0 or in the absence of infection. The role of Sp100 variants during natural HSV-1 infection remains blurry. Here, we report that Sp100A potently and independently inhibited wild-type HSV-1 and that during HSV-1 infection, cytosolic Sp100 remained stable and was increasingly secreted into the extracellular space, in association with EVs. Furthermore, the Sp100A level in secreting cells positively correlated with its level in EVs and the anti-HSV-1 potency of these EVs in recipient cells. In summary, this study implies an active antiviral role of Sp100A during wild-type HSV-1 infection and reveals a novel mechanism of Sp100A to restrict HSV-1 through extracellular communications.
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Affiliation(s)
- Yilei Ma
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Jingjing Li
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Hongchang Dong
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Zhaoxin Yang
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Lingyue Zhou
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Pei Xu
- Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China
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8
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Liao Y, Lupiani B, Reddy SM. Manipulation of Promyelocytic Leukemia Protein Nuclear Bodies by Marek's Disease Virus Encoded US3 Protein Kinase. Microorganisms 2021; 9:microorganisms9040685. [PMID: 33810320 PMCID: PMC8066686 DOI: 10.3390/microorganisms9040685] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 03/22/2021] [Accepted: 03/23/2021] [Indexed: 12/20/2022] Open
Abstract
Promyelocytic leukemia protein nuclear bodies (PML-NBs) are dynamic nuclear structures, shown to be important for herpesvirus replication; however, their role in regulating Marek’s disease virus (MDV) infection has not been studied. MDV is an oncogenic alphaherpesvirus that causes lymphoproliferative disease in chickens. MDV encodes a US3 serine/threonine protein kinase that is important for MDV replication and gene expression. In this study, we studied the role of MDV US3 in regulating PML-NBs. Using an immunofluorescence assay, we found that MDV US3 disrupts PML and SP100 in a kinase dependent manner. In addition, treatment with MG-132 (a proteasome inhibitor) could partially restore the levels of PML and SP100, suggesting that a cellular proteasome dependent degradation pathway is involved in MDV US3 induced disruption of PML and SP100. These findings provide the first evidence for the interplay between MDV proteins and PML-NBs.
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9
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Abstract
Cells activate their DNA damage response (DDR) in response to DNA virus infection, including adenoviruses, papillomaviruses, polyomaviruses, and herpesviruses. In this study, we found that the DDR kinase pathways activated in normal human fibroblasts by herpes simplex virus 1 (HSV-1) input genomic DNA, HSV-1 replicating DNA, and progeny DNA and in uninfected cells treated with etoposide are different. We also found using clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 technology that different host gene products are required for the DDR in uninfected versus infected cells. Individual DDR components can be proviral or antiviral in that ataxia-telangiectasia mutated (ATM) and p53 promote and Mre11 restricts replication of ICP0-null HSV-1, but ICP0 expression eliminates these DDR effects. Thus, in total, these results argue that HSV-1 manipulates the host cell DDR to utilize specific components for its optimal replication while inactivating the antiviral aspects of the DDR.IMPORTANCE We investigated the relationship between the DNA damage response, a collection of vital cellular pathways that repair potentially lethal damage to the genome, and the DNA virus herpes simplex virus 1. We found that infection by the virus triggers the DNA damage response, and key proteins that mediate this response have opposing effects on the replication and production of progeny viruses. Our work provides novel insights into the relationship between DNA virus infection and the cellular response to the viral genome. We speculate that viral gene products modulate this response, providing potentially novel targets for therapeutic intervention against the virus.
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10
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Herpes simplex virus 1 infection induces ubiquitination of UBE1a. Biochem J 2021; 478:261-279. [PMID: 33355669 DOI: 10.1042/bcj20200885] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Revised: 12/19/2020] [Accepted: 12/22/2020] [Indexed: 11/17/2022]
Abstract
Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.
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11
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Dogrammatzis C, Waisner H, Kalamvoki M. "Non-Essential" Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review. Viruses 2020; 13:E17. [PMID: 33374862 PMCID: PMC7824580 DOI: 10.3390/v13010017] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 12/19/2022] Open
Abstract
Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.
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Affiliation(s)
| | | | - Maria Kalamvoki
- Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA; (C.D.); (H.W.)
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12
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UBE1a Suppresses Herpes Simplex Virus-1 Replication. Viruses 2020; 12:v12121391. [PMID: 33291814 PMCID: PMC7762088 DOI: 10.3390/v12121391] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2020] [Revised: 11/30/2020] [Accepted: 12/01/2020] [Indexed: 12/22/2022] Open
Abstract
Herpes simplex virus-1 (HSV-1) is the causative agent of cold sores, keratitis, meningitis, and encephalitis. HSV-1-encoded ICP5, the major capsid protein, is essential for capsid assembly during viral replication. Ubiquitination is a post-translational modification that plays a critical role in the regulation of cellular events such as proteasomal degradation, protein trafficking, and the antiviral response and viral events such as the establishment of infection and viral replication. Ub-activating enzyme (E1, also named UBE1) is involved in the first step in the ubiquitination. However, it is still unknown whether UBE1 contributes to viral infection or the cellular antiviral response. Here, we found that UBE1a suppressed HSV-1 replication and contributed to the antiviral response. The UBE1a inhibitor PYR-41 increased HSV-1 production. Immunofluorescence analysis revealed that UBE1a highly expressing cells presented low ICP5 expression, and vice versa. UBE1a inhibition by PYR-41 and shRNA increased ICP5 expression in HSV-1-infected cells. UBE1a reduced and retarded ICP5 protein expression, without affecting transcription of ICP5 mRNA or degradation of ICP5 protein. Additionally, UBE1a interacted with ICP27, and both partially co-localized at the Hsc70 foci/virus-induced chaperone-enriched (VICE) domains. PYR-41 reduced the co-localization of UBE1a and ICP27. Thus, our findings provide insights into the mechanism of UBE1a in the cellular response to viral infection.
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Hristova DB, Lauer KB, Ferguson BJ. Viral interactions with non-homologous end-joining: a game of hide-and-seek. J Gen Virol 2020; 101:1133-1144. [PMID: 32735206 PMCID: PMC7879558 DOI: 10.1099/jgv.0.001478] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2020] [Accepted: 07/14/2020] [Indexed: 02/06/2023] Open
Abstract
There are extensive interactions between viruses and the host DNA damage response (DDR) machinery. The outcome of these interactions includes not only direct effects on viral nucleic acids and genome replication, but also the activation of host stress response signalling pathways that can have further, indirect effects on viral life cycles. The non-homologous end-joining (NHEJ) pathway is responsible for the rapid and imprecise repair of DNA double-stranded breaks in the nucleus that would otherwise be highly toxic. Whilst directly repairing DNA, components of the NHEJ machinery, in particular the DNA-dependent protein kinase (DNA-PK), can activate a raft of downstream signalling events that activate antiviral, cell cycle checkpoint and apoptosis pathways. This combination of possible outcomes results in NHEJ being pro- or antiviral depending on the infection. In this review we will describe the broad range of interactions between NHEJ components and viruses and their consequences for both host and pathogen.
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Affiliation(s)
- Dayana B. Hristova
- Department of Pathology, Division of Immunology, University of Cambridge, Cambridge, UK
| | - Katharina B. Lauer
- Department of Pathology, Division of Immunology, University of Cambridge, Cambridge, UK
- Present address: ELIXIR Hub, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Brian J. Ferguson
- Department of Pathology, Division of Immunology, University of Cambridge, Cambridge, UK
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The HSV-1 ubiquitin ligase ICP0: Modifying the cellular proteome to promote infection. Virus Res 2020; 285:198015. [PMID: 32416261 PMCID: PMC7303953 DOI: 10.1016/j.virusres.2020.198015] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 05/04/2020] [Accepted: 05/04/2020] [Indexed: 12/16/2022]
Abstract
ICP0 is a viral E3 ubiquitin ligase that promotes HSV-1 infection. ICP0 interacts with multiple component proteins of the ubiquitin pathway. ICP0 disrupts multiple cellular processes activated in response to infection ICP0 remodels the SUMO proteome to counteract host immune defences to infection. ICP0 is an attractive drug target for the development of antiviral HSV-1 therapeutics. Herpes simplex virus 1 (HSV-1) hijacks ubiquitination machinery to modify the cellular proteome to create an environment permissive for virus replication. HSV-1 encodes its own RING-finger E3 ubiquitin (Ub) ligase, Infected Cell Protein 0 (ICP0), that directly interfaces with component proteins of the Ub pathway to inactivate host immune defences and cellular processes that restrict the progression of HSV-1 infection. Consequently, ICP0 plays a critical role in the infectious cycle of HSV-1 that is required to promote the efficient onset of lytic infection and productive reactivation of viral genomes from latency. This review will describe the current knowledge regarding the biochemical properties and known substrates of ICP0 during HSV-1 infection. We will highlight the gaps in the characterization of ICP0 function and propose future areas of research required to understand fully the biological properties of this important HSV-1 regulatory protein.
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Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay. J Virol 2019; 93:JVI.00619-19. [PMID: 30996104 DOI: 10.1128/jvi.00619-19] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 04/12/2019] [Indexed: 01/23/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.
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Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0. J Virol 2018; 92:JVI.01452-18. [PMID: 30258013 DOI: 10.1128/jvi.01452-18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2018] [Accepted: 09/12/2018] [Indexed: 11/20/2022] Open
Abstract
To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.
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PUM1 is a biphasic negative regulator of innate immunity genes by suppressing LGP2. Proc Natl Acad Sci U S A 2017; 114:E6902-E6911. [PMID: 28760986 DOI: 10.1073/pnas.1708713114] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: (i) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNβ. The genes that were not activated encoded HDAC4 and NF-κB1. (ii) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. (iii) IFNβ produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. (iv) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. (v) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.
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Liu XJ, Yang B, Huang SN, Wu CC, Li XJ, Cheng S, Jiang X, Hu F, Ming YZ, Nevels M, Britt WJ, Rayner S, Tang Q, Zeng WB, Zhao F, Luo MH. Human cytomegalovirus IE1 downregulates Hes1 in neural progenitor cells as a potential E3 ubiquitin ligase. PLoS Pathog 2017; 13:e1006542. [PMID: 28750047 PMCID: PMC5549770 DOI: 10.1371/journal.ppat.1006542] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2017] [Revised: 08/08/2017] [Accepted: 07/19/2017] [Indexed: 01/12/2023] Open
Abstract
Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs). As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1) is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1) is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase. Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children, but the underlying pathogenesis of this infection remains unclear. Hes1, an important effector of Notch signaling, governs the fate of neural progenitor cells (NPCs) and fetal brain development. Here we demonstrate that: (1) HCMV infection results in loss of Hes1 protein in NPCs; (2) the HCMV immediate-early 1 protein (IE1) mediates Hes1 protein downregulation through direct interaction, which requires amino acids 451–475; (3) IE1 assembles a Hes1 ubiquitination complex and mediates Hes1 ubiquitination; and (4) IE1 also assembles an Sp100A ubiquitination complex and mediates Sp100A ubiquitination, but does not require amino acids 451–475. These results suggest that HCMV IE1 is a potential E3 ubiquitin ligase. Downregulation of Hes1 by HCMV infection and IE1 implies a novel mechanism linking Hes1 depletion to virus-induced neuropathogenesis.
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Affiliation(s)
- Xi-Juan Liu
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Bo Yang
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Sheng-Nan Huang
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Cong-Cong Wu
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Xiao-Jun Li
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Shuang Cheng
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Xuan Jiang
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- Guangzhou Institute of Pediatrics, Guangzhou Women and Children Medical Center, Guangzhou, China
| | - Fei Hu
- Wuhan Brain Hospital, Ministry of Transportation, Wuhan, Hubei, China
| | - Ying-Zi Ming
- The Third Xiangya Hospital, South Central University, Changsha, Hunan, China
| | - Michael Nevels
- School of Biology, Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife, United Kingdom
| | - William J. Britt
- Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, United States of America
| | - Simon Rayner
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- Department of Medical Genetics, Oslo University Hospital & University of Oslo, Oslo, Norway
| | - Qiyi Tang
- Department of Microbiology, Howard University College of Medicine, Howard University, Washington DC, United States of America
| | - Wen-Bo Zeng
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- * E-mail: (WBZ); (FZ); (MHL)
| | - Fei Zhao
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- * E-mail: (WBZ); (FZ); (MHL)
| | - Min-Hua Luo
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China
- University of Chinese Academy of Sciences, Beijing, China
- Guangzhou Institute of Pediatrics, Guangzhou Women and Children Medical Center, Guangzhou, China
- * E-mail: (WBZ); (FZ); (MHL)
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Andrews JL, Goodfellow FJ, Matosin N, Snelling MK, Newell KA, Huang XF, Fernandez-Enright F. Alterations of ubiquitin related proteins in the pathology and development of schizophrenia: Evidence from human and animal studies. J Psychiatr Res 2017; 90:31-39. [PMID: 28226265 DOI: 10.1016/j.jpsychires.2017.01.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2015] [Revised: 12/22/2016] [Accepted: 01/17/2017] [Indexed: 12/13/2022]
Abstract
Gene expression analyses in post-mortem schizophrenia brains suggest that a number of ubiquitin proteasome system (UPS) genes are associated with schizophrenia; however the status of UPS proteins in the schizophrenia brain is largely unknown. Ubiquitin related proteins are inherently involved in memory, neuronal survival and morphology, which are processes implicated in neurodevelopmental disorders such as schizophrenia. We examined levels of five UPS proteins (Protein Inhibitor of Activated STAT2 [PIAS2], F-Box and Leucine rich repeat protein 21 [FBXL21], Mouse Double Minute 2 homolog [MDM2], Ubiquitin Carboxyl-Terminal Hydrolase-L1 [UCHL1] and Ubiquitin Conjugating Enzyme E2D1 [UBE2D1]) involved in these neuronal processes, within the dorsolateral prefrontal cortex of post-mortem schizophrenia subjects and matched controls (n = 30/group), in addition to across neurodevelopmental time-points (juvenile, adolescent and adult stages of life), utilizing a well-established neurodevelopmental phencyclidine (PCP) animal model of schizophrenia. We observed significant reductions in PIAS2, FBXL21 and MDM2 in schizophrenia subjects compared to controls (p-values ranging from 0.002 to 0.004). In our developmental PCP model, MDM2 protein was significantly reduced in adult PCP-treated rats compared to controls (p = 0.034). Additionally, FBXL21 (p = 0.022) and UCHL1 (p = 0.022) were significantly decreased, whilst UBE2D1 was increased (p = 0.022), in juvenile phencyclidine-treated rats compared to controls. This is the first study reporting alterations of UPS proteins in post-mortem human schizophrenia subjects and in a neurodevelopmental model of schizophrenia. The findings from this study provide strong support for a role of these UPS proteins in the pathology and development of schizophrenia.
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Affiliation(s)
- Jessica L Andrews
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; Schizophrenia Research Institute, Sydney, NSW 2010, Australia.
| | - Frederic J Goodfellow
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia.
| | - Natalie Matosin
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; Schizophrenia Research Institute, Sydney, NSW 2010, Australia.
| | - Mollie K Snelling
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia.
| | - Kelly A Newell
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; Schizophrenia Research Institute, Sydney, NSW 2010, Australia.
| | - Xu-Feng Huang
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; Schizophrenia Research Institute, Sydney, NSW 2010, Australia.
| | - Francesca Fernandez-Enright
- Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; Schizophrenia Research Institute, Sydney, NSW 2010, Australia; Faculty of Social Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
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Zhang K, van Drunen Littel-van den Hurk S. Herpesvirus tegument and immediate early proteins are pioneers in the battle between viral infection and nuclear domain 10-related host defense. Virus Res 2017; 238:40-48. [DOI: 10.1016/j.virusres.2017.05.023] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 05/21/2017] [Accepted: 05/22/2017] [Indexed: 01/10/2023]
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Xu P, Roizman B. The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments. Proc Natl Acad Sci U S A 2017; 114:E3823-E3829. [PMID: 28439026 PMCID: PMC5441741 DOI: 10.1073/pnas.1703395114] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Nuclear domain 10 (ND10) bodies are small (0.1-1 μM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-β. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-β in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-β and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.
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Affiliation(s)
- Pei Xu
- Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, IL 60637
| | - Bernard Roizman
- Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, IL 60637
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Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus. J Virol 2017; 91:JVI.00006-17. [PMID: 28179534 DOI: 10.1128/jvi.00006-17] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2017] [Accepted: 02/06/2017] [Indexed: 01/30/2023] Open
Abstract
Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway.IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.
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Porter SS, Stepp WH, Stamos JD, McBride AA. Host cell restriction factors that limit transcription and replication of human papillomavirus. Virus Res 2017; 231:10-20. [PMID: 27863967 PMCID: PMC5325803 DOI: 10.1016/j.virusres.2016.11.014] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2016] [Revised: 11/09/2016] [Accepted: 11/10/2016] [Indexed: 02/08/2023]
Abstract
The life cycle of human papillomaviruses (HPV) is tightly regulated by the differentiation state of mucosal and cutaneous keratinocytes. To counteract viral infection, constitutively expressed cellular factors, which are defined herein as restriction factors, directly mitigate viral gene expression and replication. In turn, some HPV gene products target these restriction factors and abrogate their anti-viral effects to establish efficient gene expression and replication programs. Ironically, in certain circumstances, this delicate counterbalance between viral gene products and restriction factors facilitates persistent infection by HPVs. This review serves to recapitulate the current knowledge of nuclear restriction factors that directly affect the HPV infectious cycle.
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Affiliation(s)
- Samuel S Porter
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD 20892, USA; Biological Sciences Graduate Program, University of Maryland, University of Maryland, 4066 Campus Drive, College Park, MD 20742, USA
| | - Wesley H Stepp
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD 20892, USA
| | - James D Stamos
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD 20892, USA
| | - Alison A McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD 20892, USA.
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24
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Wang Z, Liu Q, Lu J, Fan P, Xie W, Qiu W, Wang F, Hu G, Zhang Y. Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing. J Biol Chem 2016; 291:26377-26387. [PMID: 27784784 DOI: 10.1074/jbc.m116.753046] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Revised: 10/17/2016] [Indexed: 11/06/2022] Open
Abstract
Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression.
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Affiliation(s)
- Ziqiang Wang
- From the School of Life Sciences, Tsinghua University, Beijing 100084, China.,the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Qing Liu
- From the School of Life Sciences, Tsinghua University, Beijing 100084, China.,the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Jinhua Lu
- Shenzhen South China Pharmaceutical Co., Ltd., Shenzhen 518055, China, and
| | - Ping Fan
- From the School of Life Sciences, Tsinghua University, Beijing 100084, China.,the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Weidong Xie
- the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Wei Qiu
- From the School of Life Sciences, Tsinghua University, Beijing 100084, China.,the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Fan Wang
- From the School of Life Sciences, Tsinghua University, Beijing 100084, China.,the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
| | - Guangnan Hu
- the Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655
| | - Yaou Zhang
- the Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China,
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25
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Xu P, Mallon S, Roizman B. PML plays both inimical and beneficial roles in HSV-1 replication. Proc Natl Acad Sci U S A 2016; 113:E3022-8. [PMID: 27162364 PMCID: PMC4889406 DOI: 10.1073/pnas.1605513113] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1-1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML(+/+) cells than in sibling PML(-/-) cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML(-/-) human cell line. We report that in PML(-/-) cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML(-/-) cells infected at 5 pfu wild-type virus per cell, but poorly in PML(-/-) cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML(-/-) infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.
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Affiliation(s)
- Pei Xu
- Marjorie B. Kovler Viral Oncology Labs, The University of Chicago, Chicago IL 60637
| | - Stephen Mallon
- Marjorie B. Kovler Viral Oncology Labs, The University of Chicago, Chicago IL 60637
| | - Bernard Roizman
- Marjorie B. Kovler Viral Oncology Labs, The University of Chicago, Chicago IL 60637
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26
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Gu H. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication. World J Virol 2016; 5:1-13. [PMID: 26870669 PMCID: PMC4735549 DOI: 10.5501/wjv.v5.i1.1] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2015] [Revised: 10/28/2015] [Accepted: 12/08/2015] [Indexed: 02/05/2023] Open
Abstract
Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host.
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27
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Heilingloh CS, Kummer M, Mühl-Zürbes P, Drassner C, Daniel C, Klewer M, Steinkasserer A. L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation. J Virol 2015; 89:11046-55. [PMID: 26311871 PMCID: PMC4621140 DOI: 10.1128/jvi.01517-15] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2015] [Accepted: 08/19/2015] [Indexed: 01/11/2023] Open
Abstract
UNLABELLED Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.
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Affiliation(s)
| | - Mirko Kummer
- Department of Immune Modulation, University Hospital Erlangen, Erlangen, Germany
| | - Petra Mühl-Zürbes
- Department of Immune Modulation, University Hospital Erlangen, Erlangen, Germany
| | - Christina Drassner
- Department of Immune Modulation, University Hospital Erlangen, Erlangen, Germany
| | - Christoph Daniel
- Department of Pathology, Nephropathology, University Hospital Erlangen, Erlangen, Germany
| | - Monika Klewer
- Department of Pathology, Nephropathology, University Hospital Erlangen, Erlangen, Germany
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28
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Boutell C, Davido DJ. A quantitative assay to monitor HSV-1 ICP0 ubiquitin ligase activity in vitro. Methods 2015; 90:3-7. [PMID: 25862948 PMCID: PMC4655872 DOI: 10.1016/j.ymeth.2015.04.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Revised: 04/02/2015] [Accepted: 04/03/2015] [Indexed: 12/21/2022] Open
Abstract
Application of near-infrared imaging to quantify ubiquitin biochemistry in vitro. A quantitative methodology to monitor E3 ubiquitin ligase activity in solution. Validation of sensitivity and dynamic linear range. Applicability to E3 ubiquitin ligase inhibitor studies. The ubiquitin–proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.
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Affiliation(s)
- Chris Boutell
- MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow G61 1QH, UK.
| | - David J Davido
- Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66049, USA
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29
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Kumari P, Narayanan S, Kumar H. Herpesviruses: interfering innate immunity by targeting viral sensing and interferon pathways. Rev Med Virol 2015; 25:187-201. [DOI: 10.1002/rmv.1836] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2014] [Revised: 03/03/2015] [Accepted: 03/04/2015] [Indexed: 12/26/2022]
Affiliation(s)
- Puja Kumari
- Laboratory of Immunology, Department of Biological Sciences; Indian Institute of Science Education and Research (IISER); Bhopal India
| | - Sathish Narayanan
- Laboratory of Virology, Department of Biological Sciences; Indian Institute of Science Education and Research (IISER); Bhopal India
| | - Himanshu Kumar
- Laboratory of Immunology, Department of Biological Sciences; Indian Institute of Science Education and Research (IISER); Bhopal India
- Laboratory of Host Defense; WPI Immunology Frontier Research Centre, Osaka University; Osaka Japan
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30
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Kennedy PGE, Rovnak J, Badani H, Cohrs RJ. A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation. J Gen Virol 2015; 96:1581-602. [PMID: 25794504 DOI: 10.1099/vir.0.000128] [Citation(s) in RCA: 92] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Herpes simplex virus type 1 (HSV-1; human herpesvirus 1) and varicella-zoster virus (VZV; human herpesvirus 3) are human neurotropic alphaherpesviruses that cause lifelong infections in ganglia. Following primary infection and establishment of latency, HSV-1 reactivation typically results in herpes labialis (cold sores), but can occur frequently elsewhere on the body at the site of primary infection (e.g. whitlow), particularly at the genitals. Rarely, HSV-1 reactivation can cause encephalitis; however, a third of the cases of HSV-1 encephalitis are associated with HSV-1 primary infection. Primary VZV infection causes varicella (chickenpox) following which latent virus may reactivate decades later to produce herpes zoster (shingles), as well as an increasingly recognized number of subacute, acute and chronic neurological conditions. Following primary infection, both viruses establish a latent infection in neuronal cells in human peripheral ganglia. However, the detailed mechanisms of viral latency and reactivation have yet to be unravelled. In both cases latent viral DNA exists in an 'end-less' state where the ends of the virus genome are joined to form structures consistent with unit length episomes and concatemers, from which viral gene transcription is restricted. In latently infected ganglia, the most abundantly detected HSV-1 RNAs are the spliced products originating from the primary latency associated transcript (LAT). This primary LAT is an 8.3 kb unstable transcript from which two stable (1.5 and 2.0 kb) introns are spliced. Transcripts mapping to 12 VZV genes have been detected in human ganglia removed at autopsy; however, it is difficult to ascribe these as transcripts present during latent infection as early-stage virus reactivation may have transpired in the post-mortem time period in the ganglia. Nonetheless, low-level transcription of VZV ORF63 has been repeatedly detected in multiple ganglia removed as close to death as possible. There is increasing evidence that HSV-1 and VZV latency is epigenetically regulated. In vitro models that permit pathway analysis and identification of both epigenetic modulations and global transcriptional mechanisms of HSV-1 and VZV latency hold much promise for our future understanding in this complex area. This review summarizes the molecular biology of HSV-1 and VZV latency and reactivation, and also presents future directions for study.
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Affiliation(s)
- Peter G E Kennedy
- 1Institute of Infection, Immunity and Inflammation, University of Glasgow, Garscube Campus, Glasgow G61 1QH, UK
| | - Joel Rovnak
- 2Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80521, USA
| | - Hussain Badani
- 3Department of Neurology, University of Colorado Medical School, Aurora, CO 80045, USA
| | - Randall J Cohrs
- 3Department of Neurology, University of Colorado Medical School, Aurora, CO 80045, USA 4Department of Microbiology, University of Colorado Medical School, Aurora, CO 80045, USA
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31
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Roizman B, Zhou G. The 3 facets of regulation of herpes simplex virus gene expression: A critical inquiry. Virology 2015; 479-480:562-7. [PMID: 25771487 DOI: 10.1016/j.virol.2015.02.036] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2014] [Revised: 02/20/2015] [Accepted: 02/20/2015] [Indexed: 11/17/2022]
Abstract
On entry into the body herpes simplex viruses (HSV) replicate in a series of steps that involves derepression of viral DNA activated by VP16, a virion protein, and sequential transcription of viral genes in a cascade fashion. HSV also enters into neurons in which viral DNA maintained as heterochromatin and with few exceptions viral gene expression is silenced. A third face of the interaction of HSV with its host cells takes place at the moment when the silenced viral genome in neurons is abruptly derepressed. The available data do no reveal evidence that HSV encodes different regulatory programs for each facet of its interaction with its host cells. Rather the data point to significant gaps in our knowledge of the mechanisms by which each facet is initiated and the roles of the infected cells at each facet of the interaction of viral gene products with the host cell.
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Affiliation(s)
- Bernard Roizman
- The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago IL 606037, United States.
| | - Guoying Zhou
- The Sino-French Hoffmann Institute of Immunology Guangzhou Medical University, Guangzhou 510182, China
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32
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Torres L, Tang Q. Immediate-Early (IE) gene regulation of cytomegalovirus: IE1- and pp71-mediated viral strategies against cellular defenses. Virol Sin 2014; 29:343-52. [PMID: 25501994 DOI: 10.1007/s12250-014-3532-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Accepted: 11/11/2014] [Indexed: 12/17/2022] Open
Abstract
Three crucial hurdles hinder studies on human cytomegalovirus (HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus-host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host (latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate-early (IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate-early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.
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Affiliation(s)
- Lilith Torres
- Department of Microbiology, Ponce Health Sciences University, Ponce Research Institute, Ponce, PR, 00716, USA
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33
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Heilingloh CS, Mühl-Zürbes P, Steinkasserer A, Kummer M. Herpes simplex virus type 1 ICP0 induces CD83 degradation in mature dendritic cells independent of its E3 ubiquitin ligase function. J Gen Virol 2014; 95:1366-1375. [PMID: 24643878 DOI: 10.1099/vir.0.062810-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Mature dendritic cells (mDCs) are the most potent antigen-presenting cells known today, as they are the only antigen-presenting cells able to induce naïve T-cells. Therefore, they play a crucial role during the induction of effective antiviral immune responses. Interestingly, the surface molecule CD83 expressed on mDCs is targeted by several viruses. As CD83 has been shown to exert co-stimulatory functions on mDCs, its downmodulation represents a viral immune escape mechanism. Mechanistically, it has been shown that herpes simplex virus type 1 infection leads to proteasomal degradation of CD83, resulting in a strongly diminished T-cell stimulatory capacity of the infected mDC. Previous data suggest that the viral immediate-early protein ICP0 (infected-cell protein 0) plays an important role in this process. In the present study, we showed that ICP0 is sufficient to induce CD83 degradation in the absence of any other viral factor. However, the mechanism of ICP0-mediated CD83 degradation is not yet understood. Here, we provide evidence that ubiquitination of lysine residues is, despite the published E3 ubiquitin ligase activity of ICP0, not necessary for CD83 degradation. This finding was underlined by the observation that expression of an ICP0 mutant lacking the E3 ubiquitin ligase domain in mDCs still induced CD83 degradation. Finally, inhibition of E1 activating enzyme using the specific inhibitor 4[4-(5-nitro-furan-2-ylmethylene)-3.5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester did not prevent CD83 degradation. Taken together, our data provide strong evidence that ICP0 alone induces CD83 degradation independent of its E3 ubiquitin ligase function and of the ubiquitin machinery.
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Affiliation(s)
- Christiane S Heilingloh
- Department of Immune Modulation, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany
| | - Petra Mühl-Zürbes
- Department of Immune Modulation, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany
| | - Alexander Steinkasserer
- Department of Immune Modulation, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany
| | - Mirko Kummer
- Department of Immune Modulation, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany
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34
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HSV-1 ICP0: An E3 Ubiquitin Ligase That Counteracts Host Intrinsic and Innate Immunity. Cells 2014; 3:438-54. [PMID: 24852129 PMCID: PMC4092860 DOI: 10.3390/cells3020438] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Accepted: 05/08/2014] [Indexed: 01/05/2023] Open
Abstract
The herpes simplex virus type 1 (HSV-1) encoded E3 ubiquitin ligase, infected cell protein 0 (ICP0), is required for efficient lytic viral replication and regulates the switch between the lytic and latent states of HSV-1. As an E3 ubiquitin ligase, ICP0 directs the proteasomal degradation of several cellular targets, allowing the virus to counteract different cellular intrinsic and innate immune responses. In this review, we will focus on how ICP0’s E3 ubiquitin ligase activity inactivates the host intrinsic defenses, such as nuclear domain 10 (ND10), SUMO, and the DNA damage response to HSV-1 infection. In addition, we will examine ICP0’s capacity to impair the activation of interferon (innate) regulatory mediators that include IFI16 (IFN γ-inducible protein 16), MyD88 (myeloid differentiation factor 88), and Mal (MyD88 adaptor-like protein). We will also consider how ICP0 allows HSV-1 to evade activation of the NF-κB (nuclear factor kappa B) inflammatory signaling pathway. Finally, ICP0’s paradoxical relationship with USP7 (ubiquitin specific protease 7) and its roles in intrinsic and innate immune responses to HSV-1 infection will be discussed.
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35
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The stability of herpes simplex virus 1 ICP0 early after infection is defined by the RING finger and the UL13 protein kinase. J Virol 2014; 88:5437-43. [PMID: 24574411 DOI: 10.1128/jvi.00542-14] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
UNLABELLED Herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a multifunctional protein that plays a key role in overcoming numerous facets of host innate immunity. A key function of ICP0 that requires an intact RING finger domain is that of an ubiquitin E3 ligase: ICP0 interacts with at least three ubiquitin-conjugating enzymes of which one, UbcH5a, is required for degradation of PML and SP100. A preceding report showed that ICP0 is highly unstable at very early times after infection but becomes stable at later times. We report here that (i) the degradation of ICP0 is not infected cell specific, (ii) the degradation does not require the interaction of ICP0 with either UbcH5a, UbcH6, or UbcH9, (iii) ICP0 is degraded both early and late in cells infected with a mutant lacking the UL13 protein kinase, (iv) ICP0 encoded by wild-type virus or the ΔUL13 mutant is stable in cells transfected with a plasmid encoding UL13 before infection, (v) ICP0 carrying mutations in the RING finger domain is stable both early and late in infection, and, finally, (vi) in cells infected with both wild type and RING finger mutant only the wild-type ICP0 is rapidly degraded at early times. The results suggest that the stability of ICP0 is mediated by the UL13 protein kinase and that the target of proteolysis is a site at or near the RING domain of ICP0. IMPORTANCE ICP0, a major regulatory protein of HSV-1, turns over rapidly early in infection but becomes stable at late times. We report that stabilization requires the presence of UL13 protein kinase and that an ICP0 with mutations in RING finger is stable. In mixed infections mutant ICP0 is stable, whereas the wild-type ICP0 is degraded. Our findings suggest that the lifestyle of HSV-1 requires an ICP0 that turns over rapidly if late proteins are absent.
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36
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HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation. Proc Natl Acad Sci U S A 2014; 111:E611-7. [PMID: 24449861 DOI: 10.1073/pnas.1323414111] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
STING (stimulator of IFN genes) activates the IFN pathway in response to cytosolic DNA. Knockout of STING in mice was reported to exacerbate the pathogenicity of herpes simplex virus 1 (HSV-1). Here we report the following: (i) STING is stable in cancer-derived HEp-2 or HeLa cells infected with wild-type HSV-1 but is degraded in cells infected with mutants lacking the genes encoding functional infected cell protein 0 (ICP0), ICP4, or the US3 protein kinase (US3-PK). In HEp-2 cells, depletion of STING by shRNA results in a decrease in the yields of wild-type or ΔICP0 viruses. (ii) STING is stable throughout infection with either wild-type or ICP0 mutant viruses in human embryonic lung cells (HEL) or HEK293T cells derived from normal tissues. In these cells, depletion of STING results in higher yields of both wild-type and ΔICP0 viruses. (iii) The US3-PK is also required for stabilization of IFI16, a nuclear DNA sensor. However, the stability of IFI16 does not correlate positively or negatively with that of STING. IFI16 is stable in STING-depleted HEL cells infected with wild-type virus. In contrast to HEL cells, IFI16 was undetectable in STING-depleted HEp-2 cells, and hence the role of HSV-1 in maintaining IFI16 could not be ascertained. The results indicate that in HSV-1-infected cells the stability of IFI16 and the function and stability of STING are dependent on cell derivation, the functional integrity of ICP0, and US3-PK, an indication that in wild-type virus-infected cells both proteins are actively stabilized. In HEp-2 cells, the stability of IFI16 requires STING.
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Seghatoleslam A, Bozorg-Ghalati F, Monabati A, Nikseresht M, Owji AA. UBE2Q1, as a Down Regulated Gene in Pediatric Acute Lymphoblastic Leukemia. INTERNATIONAL JOURNAL OF MOLECULAR AND CELLULAR MEDICINE 2014; 3:95-101. [PMID: 25035859 PMCID: PMC4082811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/23/2013] [Revised: 01/01/2014] [Accepted: 02/19/2014] [Indexed: 11/25/2022]
Abstract
Ubiquitin - proteasome system (UPS), the major protein degradation pathway in the cells, typically degrades short - lived and damaged proteins and regulates growth and stress responses. This pathway is altered in various cancers, including Acute Lymphoblastic Leukemia (ALL). ALL begins with a change in bone marrow cells and is the most common type of leukemia in children under 15 years. UBE2Q1 as a new characterized gene of E2 enzyme family is located on chromosome 1 and reported to be altered in some malignancies. In this study, we aimed to explore the expression pattern of UBE2Q1 gene in children with ALL. For this purpose, a series of RT - PCR and quantitative RT - PCR were performed on a collection of 20 bone marrow samples of ALL patients and the same number of whole blood samples of age - matched normal subjects. Gel electrophoresis of RT - PCR products revealed the expression of UBE2Q1 mRNA in most of the normal (90%) and about half of the leukemic (45%) samples. QRT - PCR data indicated that only 1 patient out of 20 (5%) showed up regulation of the gene (> 2 folds). In 4 patients (20%), the expression of UBE2Q1 mRNA was equivocal (from 1/2 to 2) and in 15 cases (75%), the gene was down regulated (> 1/2) when compared to the normal samples. In conclusion, down regulation of UBE2Q1 in the majority of the leukemic samples suggests its potential implication in the pathogenesis of ALL. UBE2Q1 can be considered as a molecular marker and a candidate targeting to treat ALL in the future.
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Affiliation(s)
- Atefeh Seghatoleslam
- Histomorphometry & Stereology Research Center, School of Medicine, Shiraz, Iran.,Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.,Corresponding author: Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
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| | - Farzaneh Bozorg-Ghalati
- Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Ahmad Monabati
- Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Mohsen Nikseresht
- Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran.
| | - Ali Akbar Owji
- Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
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Perusina Lanfranca M, Mostafa HH, Davido DJ. Two overlapping regions within the N-terminal half of the herpes simplex virus 1 E3 ubiquitin ligase ICP0 facilitate the degradation and dissociation of PML and dissociation of Sp100 from ND10. J Virol 2013; 87:13287-96. [PMID: 24089549 PMCID: PMC3838275 DOI: 10.1128/jvi.02304-13] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2013] [Accepted: 09/23/2013] [Indexed: 12/20/2022] Open
Abstract
Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in sensory neurons and can reactivate from latency under stress conditions. To promote lytic infection, the virus must interact with specific cellular factors to evade the host's antiviral defenses. The HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), activates transcription of viral genes, in part, by mediating the degradation of certain cellular proteins that play a role in host antiviral mechanisms. One component of the cellular defenses that ICP0 disrupts is the suborganelle, nuclear domain 10 (ND10), by inducing the degradation and dissociation of the major organizer of ND10, a promyelocytic leukemia (PML) and ND10 constituent, Sp100. Because previously identified domains in ICP0 explain only partially how it directs the degradation and dissociation of PML and Sp100, we hypothesized that additional regions within ICP0 may contribute to these activities, which in turn facilitate efficient viral replication. To test this hypothesis, we used a series of ICP0 truncation mutants and examined PML protein levels and PML and Sp100 immunofluorescence staining in human embryonic lung cells. Our results demonstrate that two overlapping regions within the central N-terminal portion of ICP0 (residues 212 to 311) promoted the dissociation and degradation of PML and dissociation of Sp100 (residues 212 to 427). In conclusion, we have identified two additional regions in ICP0 involved in altering ND10 antiviral defenses in a cell culture model of HSV-1 infection.
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Lin AE, Greco TM, Döhner K, Sodeik B, Cristea IM. A proteomic perspective of inbuilt viral protein regulation: pUL46 tegument protein is targeted for degradation by ICP0 during herpes simplex virus type 1 infection. Mol Cell Proteomics 2013; 12:3237-52. [PMID: 23938468 DOI: 10.1074/mcp.m113.030866] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Much like the host cells they infect, viruses must also regulate their life cycles. Herpes simples virus type 1 (HSV-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. Following entry into host cells, the virion-associated outer tegument proteins pUL46 and pUL47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. Because pUL46 has gone largely unstudied, we employed a hybrid mass spectrometry-based approach to determine how pUL46 exerts its functions during early stages of infection. For a spatio-temporal characterization of pUL46, time-lapse microscopy was performed in live cells to define its dynamic localization from 2 to 24 h postinfection. Next, pUL46-containing protein complexes were immunoaffinity purified during infection of human fibroblasts and analyzed by mass spectrometry to investigate virus-virus and virus-host interactions, as well as post-translational modifications. We demonstrated that pUL46 is heavily phosphorylated in at least 23 sites. One phosphorylation site matched the consensus 14-3-3 phospho-binding motif, consistent with our identification of 14-3-3 proteins and host and viral kinases as specific pUL46 interactions. Moreover, we determined that pUL46 specifically interacts with the viral E3 ubiquitin ligase ICP0. We demonstrated that pUL46 is partially degraded in a proteasome-mediated manner during infection, and that the catalytic activity of ICP0 is responsible for this degradation. This is the first evidence of a viral protein being targeted for degradation by another viral protein during HSV-1 infection. Together, these data indicate that pUL46 levels are tightly controlled and important for the temporal regulation of viral gene expression throughout the virus life cycle. The concept of a structural virion protein, pUL46, performing nonstructural roles is likely to reflect a theme common to many viruses, and a better understanding of these functions will be important for developing therapeutics.
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Affiliation(s)
- Aaron E Lin
- Department of Molecular Biology, Princeton University, Princeton, New Jersey
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Rivera-Molina YA, Martínez FP, Tang Q. Nuclear domain 10 of the viral aspect. World J Virol 2013; 2:110-122. [PMID: 24255882 PMCID: PMC3832855 DOI: 10.5501/wjv.v2.i3.110] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2013] [Revised: 05/31/2013] [Accepted: 07/11/2013] [Indexed: 02/05/2023] Open
Abstract
Nuclear domain 10 (ND10) are spherical bodies distributed throughout the nucleoplasm and measuring around 0.2-1.0 μm. First observed under an electron microscope, they were originally described as dense bodies found in the nucleus. They are known by a number of other names, including Promyelocytic Leukemia bodies (PML bodies), Kremer bodies, and PML oncogenic domains. ND10 are frequently associated with Cajal bodies and cleavage bodies. It has been suggested that they play a role in regulating gene transcription. ND10 were originally characterized using human autoantisera, which recognizes Speckled Protein of 100 kDa, from patients with primary biliary cirrhosis. At the immunohistochemical level, ND10 appear as nuclear punctate structures, with 10 indicating the approximate number of dots per nucleus observed. ND10 do not colocalize with kinetochores, centromeres, sites of mRNA processing, or chromosomes. Resistance of ND10 antigens to nuclease digestion and salt extraction suggest that ND10 are associated with the nuclear matrix. They are often identified by immunofluorescent assay using specific antibodies against PML, Death domain-associated protein, nuclear dot protein (NDP55), and so on. The role of ND10 has long been the subject of investigation, with the specific connection of ND10 and viral infection having been a particular focus for almost 20 years. This review summarizes the relationship of ND10 and viral infection. Some future study directions are also discussed.
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Griffiths SJ, Koegl M, Boutell C, Zenner HL, Crump CM, Pica F, Gonzalez O, Friedel CC, Barry G, Martin K, Craigon MH, Chen R, Kaza LN, Fossum E, Fazakerley JK, Efstathiou S, Volpi A, Zimmer R, Ghazal P, Haas J. A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication. PLoS Pathog 2013; 9:e1003514. [PMID: 23950709 PMCID: PMC3738494 DOI: 10.1371/journal.ppat.1003514] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2013] [Accepted: 05/24/2013] [Indexed: 11/24/2022] Open
Abstract
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.
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Affiliation(s)
| | - Manfred Koegl
- Preclinical Target Development and Genomics and Proteomics Core Facilities, German Cancer Research Center, Heidelberg, Germany
| | - Chris Boutell
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Helen L. Zenner
- Division of Virology, Department of Pathology Cambridge University, Cambridge, United Kingdom
| | - Colin M. Crump
- Division of Virology, Department of Pathology Cambridge University, Cambridge, United Kingdom
| | | | - Orland Gonzalez
- Institute for Informatics, Ludwig-Maximilians Universität München, München, Germany
| | - Caroline C. Friedel
- Institute for Informatics, Ludwig-Maximilians Universität München, München, Germany
| | - Gerald Barry
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom
| | - Kim Martin
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Marie H. Craigon
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Rui Chen
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Lakshmi N. Kaza
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Even Fossum
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - John K. Fazakerley
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom
| | - Stacey Efstathiou
- Division of Virology, Department of Pathology Cambridge University, Cambridge, United Kingdom
| | | | - Ralf Zimmer
- Institute for Informatics, Ludwig-Maximilians Universität München, München, Germany
| | - Peter Ghazal
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
- Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, United Kingdom
| | - Jürgen Haas
- Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
- Max von Pettenkofer Institut, Ludwig-Maximilians Universität München, München, Germany
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Bartocci C, Denchi EL. Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites. Front Genet 2013; 4:128. [PMID: 23847653 PMCID: PMC3705210 DOI: 10.3389/fgene.2013.00128] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2013] [Accepted: 07/14/2013] [Indexed: 11/29/2022] Open
Abstract
RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions.
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Affiliation(s)
- Cristina Bartocci
- Laboratory of Chromosome Biology and Genomic Stability, Department of Molecular and Experimental Medicine, The Scripps Research Institute La Jolla, CA, USA
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Mostafa HH, Thompson TW, Davido DJ. N-terminal phosphorylation sites of herpes simplex virus 1 ICP0 differentially regulate its activities and enhance viral replication. J Virol 2013; 87:2109-19. [PMID: 23221554 PMCID: PMC3571471 DOI: 10.1128/jvi.02588-12] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2012] [Accepted: 11/27/2012] [Indexed: 02/06/2023] Open
Abstract
The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication.
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Affiliation(s)
- Heba H Mostafa
- Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA
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HSV carrying WT REST establishes latency but reactivates only if the synthesis of REST is suppressed. Proc Natl Acad Sci U S A 2013; 110:E498-506. [PMID: 23341636 DOI: 10.1073/pnas.1222497110] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
HSVs transit from vigorous replication at the portal of entry into the body to a latent state in sensory neurons in which only noncoding (e.g., latency-associated transcript) and micro-RNAs are expressed. In productive infection, viral genes must be sequentially derepressed at two checkpoints. A leading role in the repression of viral genes is carried out by histone deacetylase (HDAC)/corepressor element-1 silencing transcription factor (CoREST)/lysinespecific demethylase1(LSD1)/RE1-silencing transcription factor (REST) repressor complex (HCLR). Previously, we reported that to define the role of the components of the HCLR complex in the establishment of latency, we constructed recombinant virus (R112) carrying a dominant-negative REST that bound response elements in DNA but could not recruit repressive proteins. This recombinant virus was unable to establish latency. In the current studies, we constructed a virus (R111) carrying WT REST with a WT genome. We report the following findings: (a) R111 readily established latent infection in trigeminal ganglia; however, although the amounts of viral DNAs in latently infected neurons were similar to those of WT virus, the levels of latency-associated transcript and micro-RNAs were 50- to 100-fold lower; (b) R111 did not spontaneously reactivate in ganglionic organ cultures; however, viral genes were expressed if the synthesis of REST was blocked by cycloheximide; and (c) histone deacetylase inhibitors reactivated the WT parent but not the R111 recombinant virus. The results suggest that REST plays a transient role in the establishment of latency but not in reactivation and suggest the existence of at least two phases at both establishment and reactivation.
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45
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Schmitz ML, Grishina I. Regulation of the tumor suppressor PML by sequential post-translational modifications. Front Oncol 2012; 2:204. [PMID: 23293771 PMCID: PMC3533183 DOI: 10.3389/fonc.2012.00204] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2012] [Accepted: 12/11/2012] [Indexed: 01/08/2023] Open
Abstract
Post-translational modifications (PTMs) regulate multiple biological functions of the promyelocytic leukemia (PML) protein and also the fission, disassembly, and rebuilding of PML nuclear bodies (PML-NBs) during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitutes typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα.
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Affiliation(s)
- M Lienhard Schmitz
- Department of Biochemistry, Medical Faculty, Justus Liebig University, German Center for Lung Research Giessen, Germany
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Boutell C, Everett RD. Regulation of alphaherpesvirus infections by the ICP0 family of proteins. J Gen Virol 2012; 94:465-481. [PMID: 23239572 DOI: 10.1099/vir.0.048900-0] [Citation(s) in RCA: 163] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is important for the regulation of lytic and latent viral infection. Like the related proteins expressed by other alphaherpesviruses, ICP0 has a zinc-stabilized RING finger domain that confers E3 ubiquitin ligase activity. This domain is essential for the core functions of ICP0 and its activity leads to the degradation of a number of cellular proteins, some of which are involved in cellular defences that restrict viral infection. The article reviews recent advances in ICP0-related research, with an emphasis on the mechanisms by which ICP0 and related proteins counteract antiviral restriction and the roles in this process of cellular nuclear substructures known as ND10 or PML nuclear bodies. We also summarize recent advances in the understanding of the biochemical aspects of ICP0 activity. These studies highlight the importance of the SUMO conjugation pathway in both intrinsic resistance to HSV-1 infection and in substrate targeting by ICP0. The topics discussed in this review are relevant not only to HSV-1 infection, but also to cellular intrinsic resistance against herpesviruses more generally and the mechanisms by which viruses can evade this restriction.
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Affiliation(s)
- Chris Boutell
- MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, Scotland, UK
| | - Roger D Everett
- MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, Scotland, UK
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Use of biotinylated plasmid DNA as a surrogate for HSV DNA to identify proteins that repress or activate viral gene expression. Proc Natl Acad Sci U S A 2012; 109:E3549-57. [PMID: 23223531 DOI: 10.1073/pnas.1218783109] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
ICP0, a key herpes simplex virus regulatory protein, functions first in the nucleus and then in the cytoplasm. The duration of its nuclear sojourn in cells transfected with DNA and then infected is related to the quantity of transfected DNA. Furthermore, ICP0 transactivates both viral genes and genes encoded by the transfected DNA. The data support the hypothesis that ICP0 is retained in the nucleus until it completes the replacement of repressive chromatin with effector proteins that enable transcription of both DNA templates.To identify the effector proteins, we transfected cells with biotinylated DNA encoding a nonviral gene and then infected the cells with wild-type virus. Proteins bound to transfected biotinylated plasmid recovered from mock-treated and infected cells were identified using mass spectrometry followed by appropriate database search. The transfected DNA from mock-infected cells yielded proteins associated with repression, whereas DNA recovered from infected cells included proteins known to enable transcription and proteins that have not been previously associated with that role. To test the hypothesis that the proteins hitherto not known to associate with viral gene expression are nevertheless essential, we tested the role of the DEAD-box helicase Ddx17. We report that Ddx17 plays a critical role in the expression of early and late viral genes. Thus, biotinylated DNA recovered from transfected infected cells can function as a surrogate for viral DNA and is a rich source of proteins that play a role in viral gene expression but which have not been previously identified in that role.
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Rivera-Molina YA, Rojas BR, Tang Q. Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression. Virol J 2012; 9:222. [PMID: 23021128 PMCID: PMC3502357 DOI: 10.1186/1743-422x-9-222] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2012] [Accepted: 09/27/2012] [Indexed: 11/29/2022] Open
Abstract
Background DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. Results Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML−/−, Daxx−/−, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. Conclusion Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.
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Affiliation(s)
- Yisel A Rivera-Molina
- Department of Microbiology/RCMI Program, Ponce School of Medicine and Health Sciences, Ponce, 00716, Puerto Rico
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Overexpression of the ubiquitin-specific protease 7 resulting from transfection or mutations in the ICP0 binding site accelerates rather than depresses herpes simplex virus 1 gene expression. J Virol 2012; 86:12871-8. [PMID: 22993145 DOI: 10.1128/jvi.01981-12] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.
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The potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1. Protein Cell 2012; 3:372-82. [PMID: 22544561 DOI: 10.1007/s13238-012-2021-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2011] [Accepted: 01/19/2012] [Indexed: 01/28/2023] Open
Abstract
Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
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