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Qiao W, Xie X, Shi PY, Ooi YS, Carette JE. Druggable genome screens identify SPP as an antiviral host target for multiple flaviviruses. Proc Natl Acad Sci U S A 2025; 122:e2421573122. [PMID: 39969998 PMCID: PMC11874179 DOI: 10.1073/pnas.2421573122] [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: 10/22/2024] [Accepted: 01/16/2025] [Indexed: 02/21/2025] Open
Abstract
Mosquito-borne flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus, and yellow fever virus, pose significant public health threats globally. Extensive efforts have led to the development of promising highly active compounds against DENV targeting viral non-structural protein 4B (NS4B) protein. However, due to the cocirculation of flaviviruses and to prepare for emerging flaviviruses, there is a need for more broadly acting antivirals. Host-directed therapy where one targets a host factor required for viral replication may be active against multiple viruses that use similar replication strategies. Here, we used a CRISPR-Cas9 library that we designed to target the druggable genome and identified signal peptide peptidase (SPP, encoded by Histocompatibility Minor 13, HM13), as a critical host factor in DENV infection. Genetic knockout or introducing mutations that disrupt the proteolytic activity of SPP markedly reduced the replication of multiple flaviviruses. Although their substrates differ, SPP has structural homology with γ-secretase, which has been pursued as a pharmacological target for Alzheimer's disease. Notably, SPP-targeting compounds exhibited potent anti-DENV activity at low nanomolar concentrations across multiple primary and disease-relevant cell types, acting specifically through SPP inhibition rather than γ-secretase inhibition. Importantly, SPP inhibitors were active at low nanomolar concentrations against flaviviruses other than DENV including ZIKV while DENV NS4B inhibitors lost activity. This study emphasizes the strong potential of SPP as a pan-flaviviral target and provides a framework for identifying host druggable targets to screen for broad-spectrum antivirals.
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Affiliation(s)
- Wenjie Qiao
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
| | - Xuping Xie
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX77555
| | - Pei-Yong Shi
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX77555
| | - Yaw Shin Ooi
- Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore169857, Singapore
- Infectious Diseases Labs, Agency for Science, Technology and Research, Singapore138648, Singapore
| | - Jan E. Carette
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
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2
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Sergejevs N, Avci D, van de Weijer ML, Corey RA, Lemberg MK, Carvalho P. Topology surveillance of the lanosterol demethylase CYP51A1 by signal peptide peptidase. J Cell Sci 2024; 137:jcs262333. [PMID: 39513424 PMCID: PMC11827857 DOI: 10.1242/jcs.262333] [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: 06/05/2024] [Accepted: 11/04/2024] [Indexed: 11/15/2024] Open
Abstract
Cleavage of transmembrane segments on target proteins by the aspartyl intramembrane protease signal peptide peptidase (SPP, encoded by HM13) has been linked to immunity, viral infection and protein quality control. How SPP recognizes its various substrates and specifies their fate remains elusive. Here, we identify the lanosterol demethylase CYP51A1 as an SPP substrate and show that SPP-catalysed cleavage triggers CYP51A1 clearance by endoplasmic reticulum-associated degradation (ERAD). We observe that SPP targets only a fraction of CYP51A1 molecules, and we identify an amphipathic helix in the CYP51A1 N terminus as a key determinant for SPP recognition. SPP recognition is remarkably specific to CYP51A1 molecules with the amphipathic helix aberrantly inserted in the membrane with a type II orientation. Thus, our data are consistent with a role for SPP in topology surveillance, triggering the clearance of certain potentially non-functional conformers.
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Affiliation(s)
- Nikita Sergejevs
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Dönem Avci
- Center for Biochemistry and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Medical Faculty, University of Cologne, 50931 Cologne, Germany
| | - Michael L. van de Weijer
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Robin A. Corey
- School of Physiology, Pharmacology & Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
| | - Marius K. Lemberg
- Center for Biochemistry and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Medical Faculty, University of Cologne, 50931 Cologne, Germany
| | - Pedro Carvalho
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
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3
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Contreras W, Groenendyk J, Gentzel M, Schönberg PY, Buchholz F, Michalak M, Schröder B, Mentrup T. Selective regulation of aspartyl intramembrane protease activity by calnexin. Cell Mol Life Sci 2024; 81:441. [PMID: 39460794 PMCID: PMC11513070 DOI: 10.1007/s00018-024-05478-8] [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: 06/12/2024] [Revised: 09/09/2024] [Accepted: 10/11/2024] [Indexed: 10/28/2024]
Abstract
Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family.
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Affiliation(s)
- Whendy Contreras
- Institute of Physiological Chemistry, Medizinische Fakultät und Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Jody Groenendyk
- Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
| | - Marc Gentzel
- Core Facility Molecular Analysis - Mass Spectrometry, Mass Spectrometry & Proteomics, Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Dresden, Germany
| | - Pascal Y Schönberg
- Medical Faculty, University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307, Dresden, Germany
| | - Frank Buchholz
- Medical Faculty, University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307, Dresden, Germany
| | - Marek Michalak
- Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
| | - Bernd Schröder
- Institute of Physiological Chemistry, Medizinische Fakultät und Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Torben Mentrup
- Institute of Physiological Chemistry, Medizinische Fakultät und Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.
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4
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Brackenridge S, John N, He W, Früh K, Borrow P, McMichael A. Regulation of the cell surface expression of classical and non-classical MHC proteins by the human cytomegalovirus UL40 and rhesus cytomegalovirus Rh67 proteins. J Virol 2024; 98:e0120624. [PMID: 39207137 PMCID: PMC11406984 DOI: 10.1128/jvi.01206-24] [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] [Received: 07/12/2024] [Accepted: 07/29/2024] [Indexed: 09/04/2024] Open
Abstract
The signal sequences of the human cytomegalovirus (CMV) UL40 protein and its rhesus CMV (RhCMV) counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/V]L, VL9) that is presented by major histocompatibility complex (MHC) antigen E (MHC-E). The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts antigen processing and presentation and MHC Ia expression. This allows infected cells to maintain MHC-E surface expression and escape killing by Natural Killer cells. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation between the two proteins. Processing of both VL9 peptides is dependent on cleavage of their signal sequences by the host protease signal peptide peptidase. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 requires only its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the transporter associated with antigen processing. Our results also suggest that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by decreasing surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of Rh67, UL40 can partially counteract the up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, and further work will be required to determine if any such differences have implications for translating a RhCMV-vectored simian immunodeficiency virus (SIV) vaccine to HIV-1 using human CMV as a vector. IMPORTANCE The protective immune response induced by a rhesus cytomegalovirus (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine in rhesus macaques depends on the presence of the viral Rh67 gene in the vaccine. The Rh67 protein contains a peptide that allows the RhCMV-infected cells to maintain expression of major histocompatibility complex (MHC) antigen E at the cell surface. We show that production of this peptide, referred to as "VL9," mirrors that of the equivalent peptide present in the human cytomegalovirus (CMV) protein UL40, despite the little sequence similarity between the two CMV proteins. We also show that the mature UL40 and Rh67 proteins, which have no previously described function, also contribute to CMV immune evasion by reducing cell surface expression of MHC proteins important for the immune system to detect infected cells. Despite these similarities, our work also reveals possible differences between Rh67 and UL40, and these may have implications for the use of human CMV as the vector for a potential HIV-1 vaccine.
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Affiliation(s)
- Simon Brackenridge
- Centre for Immuno-Oncology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Nessy John
- Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA
| | - Wanlin He
- Centre for Immuno-Oncology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Klaus Früh
- Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA
| | - Persephone Borrow
- Centre for Immuno-Oncology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Andrew McMichael
- Centre for Immuno-Oncology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
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5
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Chaudhuri D, Datta J, Majumder S, Giri K. Repurposing of drug molecules from FDA database against Hepatitis C virus E2 protein using ensemble docking approach. Mol Divers 2024; 28:1175-1188. [PMID: 37061608 DOI: 10.1007/s11030-023-10646-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Accepted: 03/31/2023] [Indexed: 04/17/2023]
Abstract
Hepatitis C virus, a member of the Flaviviridae family and genus Hepacivirus, is an enveloped, positively single stranded RNA virus. Its surface consists of a heterodimer of E1 and E2 proteins which play a crucial role in receptor binding and membrane fusion. In this study we have used in silico virtual screening by utilizing ensemble docking on the approved drugs. These drugs can bind with high efficiency to the 36 prominent conformations of the CD81 binding site clustered from a total of 3 µs MD simulation data on the E2 protein. We started with 9213 compounds from the FDA list of drugs and progressively came down to 5 compounds which have been seen to bind with very high efficiency to not only all the conformations but also the two predicted druggable pockets that encompass the CD81 binding site. MM/PBSA binding energy calculations also point to the highly stable interaction of the compounds to the E2 protein. This study may in future broaden the arsenal of therapeutics for use against HCV infection and lead to more effective care against the virus.
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Affiliation(s)
- Dwaipayan Chaudhuri
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Joyeeta Datta
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Satyabrata Majumder
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Kalyan Giri
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India.
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6
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Hsia JZ, Liu D, Haynes L, Cruz-Cosme R, Tang Q. Lipid Droplets: Formation, Degradation, and Their Role in Cellular Responses to Flavivirus Infections. Microorganisms 2024; 12:647. [PMID: 38674592 PMCID: PMC11051834 DOI: 10.3390/microorganisms12040647] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2024] [Revised: 03/18/2024] [Accepted: 03/22/2024] [Indexed: 04/28/2024] Open
Abstract
Lipid droplets (LDs) are cellular organelles derived from the endoplasmic reticulum (ER), serving as lipid storage sites crucial for maintaining cellular lipid homeostasis. Recent attention has been drawn to their roles in viral replication and their interactions with viruses. However, the precise biological functions of LDs in viral replication and pathogenesis remain incompletely understood. To elucidate the interaction between LDs and viruses, it is imperative to comprehend the biogenesis of LDs and their dynamic interactions with other organelles. In this review, we explore the intricate pathways involved in LD biogenies within the cytoplasm, encompassing the uptake of fatty acid from nutrients facilitated by CD36-mediated membranous protein (FABP/FATP)-FA complexes, and FA synthesis via glycolysis in the cytoplasm and the TCL cycle in mitochondria. While LD biogenesis primarily occurs in the ER, matured LDs are intricately linked to multiple organelles. Viral infections can lead to diverse consequences in terms of LD status within cells post-infection, potentially involving the breakdown of LDs through the activation of lipophagy. However, the exact mechanisms underlying LD destruction or accumulation by viruses remain elusive. The significance of LDs in viral replication renders them effective targets for developing broad-spectrum antivirals. Moreover, considering that reducing neutral lipids in LDs is a strategy for anti-obesity treatment, LD depletion may not pose harm to cells. This presents LDs as promising antiviral targets for developing therapeutics that are minimally or non-toxic to the host.
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Affiliation(s)
| | | | | | | | - Qiyi Tang
- Department of Microbiology, Howard University College of Medicine, Washington, DC 20059, USA; (J.Z.H.); (D.L.); (L.H.); (R.C.-C.)
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7
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Nazeer B, Khawar MB, Khalid MU, Hamid SE, Rafiq M, Abbasi MH, Sheikh N, Ali A, Fatima H, Ahmad S. Emerging role of lipophagy in liver disorders. Mol Cell Biochem 2024; 479:1-11. [PMID: 36943663 DOI: 10.1007/s11010-023-04707-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2022] [Accepted: 03/10/2023] [Indexed: 03/23/2023]
Abstract
Lipophagy is a selective degradation of lipids by a lysosomal-mediated pathway, and dysregulation of lipophagy is linked with the pathological hallmark of many liver diseases. Downregulation of lipophagy in liver cells results in abnormal accumulation of LDs (Lipid droplets) in hepatocytes which is a characteristic feature of several liver pathologies such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Contrarily, upregulation of lipophagy in activated hepatic stellate cells (HSCs) is associated with hepatic fibrosis and cirrhosis. Lipid metabolism reprogramming in violent cancer cells contributes to the progression of liver cancer. In this review, we have summarized the recent studies focusing on various components of the lipophagic machinery that can be modulated for their potential role as therapeutic agents against a wide range of liver diseases.
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Affiliation(s)
- Bismillah Nazeer
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
| | - Muhammad Babar Khawar
- Applied Molecular Biology and Biomedicine Lab, Department of Zoology, University of Narowal, Narowal, Pakistan.
| | - Muhammad Usman Khalid
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
| | - Syeda Eisha Hamid
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
| | - Mussarat Rafiq
- Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan
| | | | - Nadeem Sheikh
- Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan.
| | - Ahmad Ali
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
| | - Hooriya Fatima
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
| | - Sadia Ahmad
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan
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8
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Mentrup T, Leinung N, Patel M, Fluhrer R, Schröder B. The role of SPP/SPPL intramembrane proteases in membrane protein homeostasis. FEBS J 2024; 291:25-44. [PMID: 37625440 DOI: 10.1111/febs.16941] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Revised: 07/03/2023] [Accepted: 08/23/2023] [Indexed: 08/27/2023]
Abstract
Signal peptide peptidase (SPP) and the four SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo-lysosomal system. Despite individual cleavage characteristics, they all cleave single-span transmembrane proteins with a type II orientation exhibiting a cytosolic N-terminus. Though the identification of substrates is not complete, SPP/SPPL-mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)-physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho-physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge.
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Affiliation(s)
- Torben Mentrup
- Institute for Physiological Chemistry, Technische Universität Dresden, Germany
| | - Nadja Leinung
- Institute for Physiological Chemistry, Technische Universität Dresden, Germany
| | - Mehul Patel
- Institute for Physiological Chemistry, Technische Universität Dresden, Germany
| | - Regina Fluhrer
- Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Germany
- Center for Interdisciplinary Health Research, University of Augsburg, Germany
| | - Bernd Schröder
- Institute for Physiological Chemistry, Technische Universität Dresden, Germany
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9
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Höppner S, Schröder B, Fluhrer R. Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling. FEBS J 2023; 290:5456-5474. [PMID: 37786993 DOI: 10.1111/febs.16968] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 09/13/2023] [Accepted: 09/29/2023] [Indexed: 10/04/2023]
Abstract
More than 20 years ago, signal peptide peptidase (SPP) and its homologues, the signal peptide peptidase-like (SPPL) proteases have been identified based on their sequence similarity to presenilins, a related family of intramembrane aspartyl proteases. Other than those for the presenilins, no high-resolution structures for the SPP/SPPL proteases are available. Despite this limitation, over the years bioinformatical and biochemical data have accumulated, which altogether have provided a picture of the overall structure and topology of these proteases, their localization in the cell, the process of substrate recognition, their cleavage mechanism, and their function. Recently, the artificial intelligence-based structure prediction tool AlphaFold has added high-confidence models of the expected fold of SPP/SPPL proteases. In this review, we summarize known structural aspects of the SPP/SPPL family as well as their substrates. Of particular interest are the emerging substrate recognition and catalytic mechanisms that might lead to the prediction and identification of more potential substrates and deeper insight into physiological and pathophysiological roles of proteolysis.
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Affiliation(s)
- Sabine Höppner
- Biochemistry and Molecular Biology, Faculty of Medicine, Institute of Theoretical Medicine, University of Augsburg, Germany
| | - Bernd Schröder
- Institute for Physiological Chemistry, Technische Universität Dresden, Germany
| | - Regina Fluhrer
- Biochemistry and Molecular Biology, Faculty of Medicine, Institute of Theoretical Medicine, University of Augsburg, Germany
- Center for Interdisciplinary Health Research, University of Augsburg, Germany
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10
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Wu Y, Thomas GM, Thomsen M, Bahri S, Lieberman RL. Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro. J Biol Chem 2023; 299:105401. [PMID: 38270390 PMCID: PMC10679502 DOI: 10.1016/j.jbc.2023.105401] [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: 07/11/2023] [Revised: 09/12/2023] [Accepted: 10/11/2023] [Indexed: 01/26/2024] Open
Abstract
Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-β plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.
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Affiliation(s)
- Yuqi Wu
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Gwendell M Thomas
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Max Thomsen
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Sara Bahri
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Raquel L Lieberman
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA.
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11
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Abstract
The steatotic diseases of metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), and chronic hepatitis C (HCV) account for the majority of liver disease prevalence, morbidity, and mortality worldwide. While these diseases have distinct pathogenic and clinical features, dysregulated lipid droplet (LD) organelle biology represents a convergence of pathogenesis in all three. With increasing understanding of hepatocyte LD biology, we now understand the roles of LD proteins involved in these diseases but also how genetics modulate LD biology to either exacerbate or protect against the phenotypes associated with steatotic liver diseases. Here, we review the history of the LD organelle and its biogenesis and catabolism. We also review how this organelle is critical not only for the steatotic phenotype of liver diseases but also for their advanced phenotypes. Finally, we summarize the latest attempts and challenges of leveraging LD biology for therapeutic gain in steatotic diseases. In conclusion, the study of dysregulated LD biology may lead to novel therapeutics for the prevention of disease progression in the highly prevalent steatotic liver diseases of MASLD, ALD, and HCV.
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Affiliation(s)
- Joseph L Dempsey
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of Washington, Seattle, Washington
| | - George N Ioannou
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of Washington, Seattle, Washington
- Division of Gastroenterology, Veterans Affairs Puget Sound Healthcare System Seattle, Washington
| | - Rotonya M Carr
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of Washington, Seattle, Washington
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12
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Shen HH, Zhao Q, Wen YP, Wu R, Du SY, Huang XB, Wen XT, Cao SJ, Zeng L, Yan QG. Porcine reproductive and respiratory syndrome virus upregulates SMPDL3B to promote viral replication by modulating lipid metabolism. iScience 2023; 26:107450. [PMID: 37583552 PMCID: PMC10424083 DOI: 10.1016/j.isci.2023.107450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 06/04/2023] [Accepted: 07/17/2023] [Indexed: 08/17/2023] Open
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat to the health of pigs globally. Host factors play a critical role in PRRSV replication. Using PRRSV as a model for genome-scale CRISPR knockout (KO) screening, we identified a host factor critical to PRRSV infection: sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Our findings show that SMPDL3B restricted PRRSV attachment, entry, replication, and secretion and that its depletion significantly inhibited PRRSV proliferation, indicating that SMPDL3B plays a positive role in PRRSV replication. Our data also show that SMPDL3B deficiency resulted in an accumulation of intracellular lipid droplets (LDs). The expression level of key genes (ACC, SCD-1, and FASN) involved in lipogenesis was increased, whereas the fundamental lipolysis gene, ATGL, was inhibited when SMPDL3B was knocked down. Overall, our findings suggest that SMPDL3B deficiency can effectively inhibit viral infection through the modulation of lipid metabolism.
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Affiliation(s)
- Huan-Huan Shen
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Qin Zhao
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Yi-Ping Wen
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Rui Wu
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Sen-Yan Du
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Xiao-Bo Huang
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Xin-Tian Wen
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - San-Jie Cao
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
| | - Lei Zeng
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan Province, China
| | - Qi-Gui Yan
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 610000, Sichuan Province, China
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13
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Itell HL, Humes D, Overbaugh J. Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4 + T cells. Cell Rep 2023; 42:112556. [PMID: 37227817 PMCID: PMC10592456 DOI: 10.1016/j.celrep.2023.112556] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Revised: 03/30/2023] [Accepted: 05/05/2023] [Indexed: 05/27/2023] Open
Abstract
Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4+ T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4+ T cells by performing CRISPR-knockout screens with a custom library that specifically targets ISGs expressed in CD4+ T cells. Our investigation identifies previously undescribed HIV-restricting ISGs (HM13, IGFBP2, LAP3) and finds that two factors characterized in other HIV infection models (IFI16 and UBE2L6) mediate IFN restriction in T cells. Inactivation of these five ISGs in combination further diminishes IFN's protective effect against diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.
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Affiliation(s)
- Hannah L Itell
- Molecular and Cellular Biology PhD Program, University of Washington, Seattle, WA 98195, USA; Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA
| | - Daryl Humes
- Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA
| | - Julie Overbaugh
- Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA.
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14
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Collett S, Earnest L, Carrera Montoya J, Edeling MA, Yap A, Wong CY, Christiansen D, Roberts J, Mumford J, Lecouturier V, Pavot V, Marco S, Loi JK, Simmons C, Gulab SA, Mackenzie JM, Elbourne A, Ramsland PA, Cameron G, Hans D, Godfrey DI, Torresi J. Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates. Front Microbiol 2023; 14:1065609. [PMID: 37350788 PMCID: PMC10282183 DOI: 10.3389/fmicb.2023.1065609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Accepted: 05/17/2023] [Indexed: 06/24/2023] Open
Abstract
The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.
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Affiliation(s)
- Simon Collett
- School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC, Australia
| | - Linda Earnest
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Julio Carrera Montoya
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Melissa A. Edeling
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Ashley Yap
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Chinn Yi Wong
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Dale Christiansen
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Jason Roberts
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
- Department of Infectious Diseases, The University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
| | - Jamie Mumford
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
| | | | | | | | - Joon Keit Loi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Cameron Simmons
- Institute of Vector-Borne Disease, Monash University, Clayton, VIC, Australia
| | - Shivali A. Gulab
- Avalia Immunotherapies Limited, Wellington, New Zealand
- Vaccine Alliance Aotearoa New Zealand, Wellington, New Zealand
| | - Jason M. Mackenzie
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Aaron Elbourne
- School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC, Australia
| | - Paul A. Ramsland
- School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC, Australia
- Department of Surgery Austin Health, University of Melbourne, Heidelberg, VIC, Australia
- Department of Immunology, Central Clinical School, Monash University, Melbourne, VIC, Australia
| | - Garth Cameron
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Dhiraj Hans
- Research, Innovation and Commercialisation, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
| | - Dale I. Godfrey
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
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15
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Contreras W, Bazan JF, Mentrup T. The transmembrane domain of Frey1 harbors a transplantable inhibitory motif for intramembrane proteases. Cell Mol Life Sci 2023; 80:170. [PMID: 37261541 DOI: 10.1007/s00018-023-04823-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 05/23/2023] [Accepted: 05/23/2023] [Indexed: 06/02/2023]
Abstract
Although aspartic intramembrane-cleaving proteases (I-CLIPs) are crucial switches of multiple signaling pathways and involved in several devastating diseases, little is known about their physiological regulation. We have recently identified Frey regulator of sperm-oocyte fusion 1 (Frey1) as an inhibitory protein of Signal Peptide Peptidase-like 2c (SPPL2c), a member of this protease family. Employing structure modeling along with cell-based inhibition and interaction studies, we identify a short motif within the Frey1 transmembrane domain essential for inhibition of SPPL2c. Intriguingly, this motif can be transplanted to the SPPL2c substrate PLN, thereby transforming it into an inhibitor of this enzyme. It can be adopted for the generation of Notch1-based γ-Secretase inhibitors demonstrating its versatile use among aspartic I-CLIPs. In summary, we describe a mechanism of aspartic I-CLIP inhibition which allows the targeted generation of specific inhibitors of these enzymes and might enable the identification of endogenous negative regulators of these enzymes.
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Affiliation(s)
- Whendy Contreras
- Institute of Physiological Chemistry, Technische Universität Dresden, Fiedlerstraße 42, 01307, Dresden, Germany
| | - J Fernando Bazan
- Unit for Structural Biology, VIB-UGent Center for Inflammation Research, Ghent, Belgium
| | - Torben Mentrup
- Institute of Physiological Chemistry, Technische Universität Dresden, Fiedlerstraße 42, 01307, Dresden, Germany.
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16
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Itell HL, Humes D, Overbaugh J. Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4 + T cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.07.527545. [PMID: 36798236 PMCID: PMC9934674 DOI: 10.1101/2023.02.07.527545] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/10/2023]
Abstract
Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4 + T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4 + T cells. We performed CRISPR-knockout screens using a custom library that specifically targets ISGs expressed in CD4 + T cells and validated top hits. Our investigation identified new HIV-restricting ISGs (HM13, IGFBP2, LAP3) and found that two previously studied factors (IFI16, UBE2L6) are IFN effectors in T cells. Inactivation of these five ISGs in combination further diminished IFN’s protective effect against six diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.
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Affiliation(s)
- Hannah L. Itell
- Molecular and Cellular Biology PhD Program, University of Washington, Seattle, WA, 98109, USA
- Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA, 98109, USA
| | - Daryl Humes
- Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA, 98109, USA
- Present address: Tr1X Inc, La Jolla, CA, 92037, USA
| | - Julie Overbaugh
- Division of Human Biology, Fred Hutchinson Cancer Center, Seattle, WA, 98109, USA
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17
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Diaz O, Vidalain PO, Ramière C, Lotteau V, Perrin-Cocon L. What role for cellular metabolism in the control of hepatitis viruses? Front Immunol 2022; 13:1033314. [PMID: 36466918 PMCID: PMC9713817 DOI: 10.3389/fimmu.2022.1033314] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 11/02/2022] [Indexed: 11/26/2023] Open
Abstract
Hepatitis B, C and D viruses (HBV, HCV, HDV, respectively) specifically infect human hepatocytes and often establish chronic viral infections of the liver, thus escaping antiviral immunity for years. Like other viruses, hepatitis viruses rely on the cellular machinery to meet their energy and metabolite requirements for replication. Although this was initially considered passive parasitism, studies have shown that hepatitis viruses actively rewire cellular metabolism through molecular interactions with specific enzymes such as glucokinase, the first rate-limiting enzyme of glycolysis. As part of research efforts in the field of immunometabolism, it has also been shown that metabolic changes induced by viruses could have a direct impact on the innate antiviral response. Conversely, detection of viral components by innate immunity receptors not only triggers the activation of the antiviral defense but also induces in-depth metabolic reprogramming that is essential to support immunological functions. Altogether, these complex triangular interactions between viral components, innate immunity and hepatocyte metabolism may explain why chronic hepatitis infections progressively lead to liver inflammation and progression to cirrhosis, fibrosis and hepatocellular carcinoma (HCC). In this manuscript, we first present a global overview of known connections between the innate antiviral response and cellular metabolism. We then report known molecular mechanisms by which hepatitis viruses interfere with cellular metabolism in hepatocytes and discuss potential consequences on the innate immune response. Finally, we present evidence that drugs targeting hepatocyte metabolism could be used as an innovative strategy not only to deprive viruses of key metabolites, but also to restore the innate antiviral response that is necessary to clear infection.
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Affiliation(s)
- Olivier Diaz
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Pierre-Olivier Vidalain
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Christophe Ramière
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France
| | - Vincent Lotteau
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
| | - Laure Perrin-Cocon
- CIRI, Centre International de Recherche en Infectiologie, Team VIRal Infection, Metabolism and Immunity, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France
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18
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Hochman J, Braitbard O. Life after Cleavage: The Story of a β-Retroviral (MMTV) Signal Peptide-From Murine Lymphoma to Human Breast Cancer. Viruses 2022; 14:v14112435. [PMID: 36366533 PMCID: PMC9694287 DOI: 10.3390/v14112435] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Revised: 10/31/2022] [Accepted: 10/31/2022] [Indexed: 11/06/2022] Open
Abstract
An increasing body of evidence in recent years supports an association of the betaretrovirus mouse mammary tumor virus (MMTV) with human breast cancer. This is an issue that still raises heated controversy. We have come to address this association using the signal peptide p14 of the MMTV envelope precursor protein as a key element of our strategy. In addition to its signal peptide function, p14 has some significant post endoplasmic reticulum (ER)-targeting characteristics: (1) it localizes to nucleoli where it binds key proteins (RPL5 and B23) involved (among other activities) in the regulation of nucleolar stress response, ribosome biogenesis and p53 stabilization; (2) p14 is a nuclear export factor; (3) it is expressed on the cell surface of infected cells, and as such, is amenable to, and successfully used, in preventive vaccination against experimental tumors that harbor MMTV; (4) the growth of such tumors is impaired in vivo using a combination of monoclonal anti-p14 antibodies or adoptive T-cell transfer treatments; (5) p14 is a phospho-protein endogenously phosphorylated by two different serine kinases. The phosphorylation status of the two sites determines whether p14 will function in an oncogenic or tumor-suppressing capacity; (6) transcriptional activation of genes (RPL5, ErbB4) correlates with the oncogenic potential of MMTV; (7) finally, polyclonal anti-p14 antibodies have been applied in immune histochemistry analyses of breast cancer cases using formalin fixed paraffin-embedded sections, supporting the associations of MMTV with the disease. Taken together, the above findings constitute a road map towards the diagnosis and possible prevention and treatment of MMTV-associated breast cancer.
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Affiliation(s)
- Jacob Hochman
- Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
- Correspondence: ; Tel.: +972-54-441-4370
| | - Ori Braitbard
- Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
- Department of Bioinformatics, The Faculty of Life and Health Sciences, Jerusalem College of Technology, Jerusalem 9372115, Israel
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19
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Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLoS Pathog 2022; 18:e1010895. [PMID: 36215335 PMCID: PMC9616216 DOI: 10.1371/journal.ppat.1010895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 10/28/2022] [Accepted: 09/25/2022] [Indexed: 11/16/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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20
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Zhang G, Lv X, Yang Q, Liu H. Identification of HM13 as a prognostic indicator and a predictive biomarker for immunotherapy in hepatocellular carcinoma. BMC Cancer 2022; 22:888. [PMID: 35964022 PMCID: PMC9375928 DOI: 10.1186/s12885-022-09987-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Accepted: 08/02/2022] [Indexed: 12/12/2022] Open
Abstract
Background Histocompatibility minor 13 (HM13) is a signal sequence stubbed intramembrane cleavage catalytic protein that is essential for cell signaling, intracellular communication, and cancer. However, the expression of HM13 and its prognostic value, association with tumor-infiltrating immune cells (TIICs) in the microenvironment, and potential to predict immunotherapeutic response in HCC are unknown. Methods The HM13 expression, clinicopathology analysis, and its influence on survival were analyzed in multiple public databases and further verified in collected HCC and normal tissues by qRT-PCR and immunohistochemistry staining assay (IHC). Furthermore, the lentivirus vector encoding HM13-shRNA to manipulate HM13 expression was selected to investigate whether HM13 could influence the malignant growth and metastasis potential of HCC cells. Finally, significant impacts of HM13 on the HCC tumor microenvironment (TME) and reaction to immune checkpoint inhibitors were analyzed. Results Upregulated HM13 was substantially correlated with poor prognosis in patients with HCC, and could facilitate the proliferation and migratory potential of HCC cells. Additionally, patients with high HM13 expression might be more sensitive to immunotherapy. Conclusions HM13 might be a prognostic biomarker and potential molecular therapeutic target for HCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09987-2.
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Affiliation(s)
- Genhao Zhang
- Department of Blood transfusion, Zhengzhou University First Affiliated Hospital, Zhengzhou, China
| | - Xianping Lv
- Department of Blood transfusion, Zhengzhou University First Affiliated Hospital, Zhengzhou, China
| | - Qiankun Yang
- Department of Blood transfusion, Zhengzhou University First Affiliated Hospital, Zhengzhou, China
| | - Hongchun Liu
- Department of Medical Laboratory, Zhengzhou University First Affiliated Hospital, Zhengzhou, China.
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21
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Devi P, Punga T, Bergqvist A. Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles. Viruses 2022; 14:v14040761. [PMID: 35458491 PMCID: PMC9031069 DOI: 10.3390/v14040761] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 03/31/2022] [Accepted: 04/03/2022] [Indexed: 02/05/2023] Open
Abstract
Hepatitis C virus (HCV) is the primary pathogen responsible for liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been linked to several aspects of HCV pathology, including oncogenesis, immune evasion and stress responses. We and others have previously shown that C expression in various cell lines activates Ca2+ signaling and alters Ca2+ homeostasis. In this study, we identified two distinct C protein regions that are required for the activation of Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in self-association of C, amino acids 1–68 were critical for NFAT activation. Sedimentation analysis of four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain was not required for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein to function. Our results indicate that targeting of HCV C to the ER is necessary but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.
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Affiliation(s)
- Priya Devi
- Department of Medical Sciences, Uppsala University, SE 75185 Uppsala, Sweden;
| | - Tanel Punga
- Department of Medical Biochemistry and Microbiology, Uppsala University, SE 75123 Uppsala, Sweden;
| | - Anders Bergqvist
- Department of Medical Sciences, Uppsala University, SE 75185 Uppsala, Sweden;
- Clinical Microbiology and Hospital Infection Control, Uppsala University Hospital, SE 75185 Uppsala, Sweden
- Correspondence: ; Tel.: +46-186113937
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22
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Schwake C, Hyon M, Chishti AH. Signal peptide peptidase: A potential therapeutic target for parasitic and viral infections. Expert Opin Ther Targets 2022; 26:261-273. [PMID: 35235480 DOI: 10.1080/14728222.2022.2047932] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
INTRODUCTION Signal peptide peptidase (SPP) is a GxGD-type intramembrane-cleaving aspartyl protease responsible for clearing accumulating signal peptides in the endoplasmic reticulum. SPP is conserved among all kingdoms and is essential for maintaining cell homeostasis. Inhibition of SPP with selective inhibitors and the structurally similar HIV protease inhibitors results in signal peptide accumulation and subsequent cell death. Identification of SPP homologues in major human parasitic infections has opened a new therapeutic opportunity. Moreover, the essentiality of mammalian SPP-mediated viral protein processing during infection is emerging. AREAS COVERED This review introduces the discovery and biological function of human SPP enzymes and identify parasitic homologues as pharmacological targets of both SPP and HIV protease inhibitors. Later, the role of mammalian SPP during viral infection and how disruption of host SPP can be employed as a novel antiviral therapy are examined and discussed. EXPERT OPINION Parasitic and viral infections cause severe health and economic burden, exacerbated by the lack of new therapeutics in the pipeline. SPP has been shown to be essential for malaria parasite growth and encouraging evidence in other parasites demonstrates broad essentiality of these proteases as therapeutic targets. As drug resistant parasite and viruses emerge, SPP inhibition will provide a new generation of compounds to counter the growing threat of antimicrobial resistance.
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Affiliation(s)
- Christopher Schwake
- Department of Developmental, Molecular, and Chemical Biology, Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA
| | - Michael Hyon
- Department of Developmental, Molecular, and Chemical Biology, Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA
| | - Athar H Chishti
- Department of Developmental, Molecular, and Chemical Biology, Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA
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23
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Alzahrani N, Wu MJ, Sousa CF, Kalinina OV, Welsch C, Yi M. SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency. PLoS Pathog 2022; 18:e1010310. [PMID: 35130329 PMCID: PMC8853643 DOI: 10.1371/journal.ppat.1010310] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 02/17/2022] [Accepted: 01/26/2022] [Indexed: 11/18/2022] Open
Abstract
Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.
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Affiliation(s)
- Nabeel Alzahrani
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Ming-Jhan Wu
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Carla F. Sousa
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
| | - Olga V. Kalinina
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
- Medical Faculty, Saarland University, Homburg, Germany
| | - Christoph Welsch
- Department of Internal Medicine 1, Goethe University Hospital, Frankfurt am Main, Germany
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
- * E-mail:
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Wang S, Jaggi U, Tormanen K, Hirose S, Ghiasi H. Absence of signal peptide peptidase in peripheral sensory neurons affects latency-reactivation in HSV-1 ocularly infected mice. PLoS Pathog 2022; 18:e1010281. [PMID: 35100323 PMCID: PMC8830783 DOI: 10.1371/journal.ppat.1010281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 02/10/2022] [Accepted: 01/17/2022] [Indexed: 12/05/2022] Open
Abstract
We previously reported that HSV-1 infectivity in vitro and in vivo requires HSV glycoprotein K (gK) binding to the ER signal peptide peptidase (SPP). Anterograde-retrograde transport via peripheral nerves between the site of infection (i.e., eye) and the site of latency (neurons) is a critical process to establish latency and subsequent viral reactivation. Given the essential role of neurons in HSV-1 latency-reactivation, we generated mice lacking SPP specifically in peripheral sensory neurons by crossing Advillin-Cre mice with SPPfl/fl mice. Expression of SPP mRNA and protein were significantly lower in neurons of Avil-SPP-/- mice than in control mice despite similar levels of HSV-1 replication in the eyes of Avil-SPP-/- mice and control mice. Viral transcript levels in isolated neurons of infected mice on days 2 and 5 post infection were lower than in control mice. Significantly less LAT, gB, and PD-1 expression was seen during latency in isolated neurons and total trigeminal ganglia (TG) of Avil-SPP-/- mice than in control mice. Finally, reduced latency and reduced T cell exhaustion in infected Avil-SPP-/- mice correlated with slower and no reactivation. Overall, our results suggest that blocking SPP expression in peripheral sensory neurons does not affect primary virus replication or eye disease but does reduce latency-reactivation. Thus, blocking of gK binding to SPP may be a useful tool to reduce latency-reactivation. HSV-1 gK and the ER protein SPP are both essential and highly conserved proteins. Their interaction is important for virus infectivity in vitro and in vivo. To evaluate the importance of gK binding to SPP in the peripheral nervous system, we generated SPP conditional knockout mice in peripheral nervous system using Advillin-Cre mice. The absence of SPP in peripheral nervous system significantly reduced latency-reactivation as well as T cell exhaustion.
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Affiliation(s)
- Shaohui Wang
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Ujjaldeep Jaggi
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Kati Tormanen
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Satoshi Hirose
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Homayon Ghiasi
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
- * E-mail:
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25
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Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering. Methods Mol Biol 2021. [PMID: 33877630 DOI: 10.1007/978-1-0716-1394-8_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370-377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.
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26
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Stephenson RA, Thomalla JM, Chen L, Kolkhof P, White RP, Beller M, Welte MA. Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones. Development 2021; 148:271212. [PMID: 34355743 DOI: 10.1242/dev.199381] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Accepted: 07/05/2021] [Indexed: 12/15/2022]
Abstract
Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.
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Affiliation(s)
- Roxan A Stephenson
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | | | - Lili Chen
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | - Petra Kolkhof
- Institute for Mathematical Modeling of Biological Systems, Systems Biology of Lipid Metabolism, Heinrich Heine University Düsseldorf, Düsseldorf 40225, Germany
| | - Roger P White
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | - Mathias Beller
- Institute for Mathematical Modeling of Biological Systems, Systems Biology of Lipid Metabolism, Heinrich Heine University Düsseldorf, Düsseldorf 40225, Germany
| | - Michael A Welte
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
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Wang S, Jaggi U, Yu J, Ghiasi H. Blocking HSV-1 glycoprotein K binding to signal peptide peptidase reduces virus infectivity in vitro and in vivo. PLoS Pathog 2021; 17:e1009848. [PMID: 34352042 PMCID: PMC8370620 DOI: 10.1371/journal.ppat.1009848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Revised: 08/17/2021] [Accepted: 07/28/2021] [Indexed: 11/19/2022] Open
Abstract
HSV glycoprotein K (gK) is an essential herpes protein that contributes to enhancement of eye disease. We previously reported that gK binds to signal peptide peptidase (SPP) and that depletion of SPP reduces HSV-1 infectivity in vivo. To determine the therapeutic potential of blocking gK binding to SPP on virus infectivity and pathogenicity, we mapped the gK binding site for SPP to a 15mer peptide within the amino-terminus of gK. This 15mer peptide reduced infectivity of three different virus strains in vitro as determined by plaque assay, FACS, and RT-PCR. Similarly, the 15mer peptide reduced ocular virus replication in both BALB/c and C57BL/6 mice and also reduced levels of latency and exhaustion markers in infected mice when compared with control treated mice. Addition of the gK-15mer peptide also increased the survival of infected mice when compared with control mice. These results suggest that blocking gK binding to SPP using gK peptide may have therapeutic potential in treating HSV-1-associated infection. Signal peptide peptidase (SPP) and HSV-1 glycoprotein K (gK) are essential genes in the host and virus, respectively. SPP and gK genes are both highly conserved. Previously we reported that gK binding to SPP is important for virus infectivity in vitro and in vivo. In this study we have identified the gK binding site to SPP and have shown that a gK peptide that blocks gK binding to SPP can block HSV-1 infectivity in vitro and in vivo using different strains of virus and mice. Thus, the ability of this peptide to block gK binding to SPP may be a useful tool to control HSV-1-induced eye disease in patients with herpes stromal keratitis (HSK).
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Affiliation(s)
- Shaohui Wang
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Ujjaldeep Jaggi
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Jack Yu
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Homayon Ghiasi
- Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
- * E-mail:
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Li HC, Yang CH, Lo SY. Cellular factors involved in the hepatitis C virus life cycle. World J Gastroenterol 2021; 27:4555-4581. [PMID: 34366623 PMCID: PMC8326260 DOI: 10.3748/wjg.v27.i28.4555] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/04/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 970, Taiwan
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan
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Gallardo-Flores CE, Colpitts CC. Cyclophilins and Their Roles in Hepatitis C Virus and Flavivirus Infections: Perspectives for Novel Antiviral Approaches. Pathogens 2021; 10:902. [PMID: 34358052 PMCID: PMC8308494 DOI: 10.3390/pathogens10070902] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 07/07/2021] [Accepted: 07/15/2021] [Indexed: 12/19/2022] Open
Abstract
Cyclophilins are cellular peptidyl-prolyl isomerases that play an important role in viral infections, with demonstrated roles in the replication of hepatitis C virus (HCV) and other viruses in the Flaviviridae family, such as dengue virus (DENV) and yellow fever virus (YFV). Here, we discuss the roles of cyclophilins in HCV infection and provide a comprehensive overview of the mechanisms underlying the requirement for cyclophilins during HCV replication. Notably, cyclophilin inhibitor therapy has been demonstrated to be effective in reducing HCV replication in chronically infected patients. While the roles of cyclophilins are relatively well-understood for HCV infection, cyclophilins are more recently emerging as host factors for flavivirus infection as well, providing potential new therapeutic avenues for these viral infections which currently lack antiviral therapies. However, further studies are required to elucidate the roles of cyclophilins in flavivirus replication. Here, we review the current knowledge of the role of cyclophilins in HCV infection to provide a conceptual framework to understand how cyclophilins may contribute to other viral infections, such as DENV and YFV. Improved understanding of the roles of cyclophilins in viral infection may open perspectives for the development of cyclophilin inhibitors as effective antiviral therapeutics for HCV and related viruses.
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Affiliation(s)
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada;
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30
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The E rns Carboxyterminus: Much More Than a Membrane Anchor. Viruses 2021; 13:v13071203. [PMID: 34201636 PMCID: PMC8310223 DOI: 10.3390/v13071203] [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: 05/06/2021] [Revised: 06/15/2021] [Accepted: 06/17/2021] [Indexed: 12/12/2022] Open
Abstract
Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.
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31
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Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells. PLoS Pathog 2021; 17:e1009496. [PMID: 33872335 PMCID: PMC8084336 DOI: 10.1371/journal.ppat.1009496] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 04/29/2021] [Accepted: 03/23/2021] [Indexed: 12/17/2022] Open
Abstract
LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules. Members of the Long Interspersed Nuclear Element 1 (LINE-1, L1) class of retrotransposons account for ~17% of the human genome and include ~100–150 intact L1 loci that are still functional. L1 mobilization is known to affect genomic integrity, thereby leading to disease-causing mutations, but little is known about the impact of exogenous viral infections on L1 and vice versa. While L1 retrotransposition is controlled by various mechanisms including CpG methylation, hypomethylation of L1 has been observed in hepatocellular carcinoma tissues of hepatitis C virus (HCV)-infected patients. Here, we demonstrate molecular interactions between HCV and L1 elements. HCV infection stably increases cellular levels of the L1-encoded ORF1 protein (L1ORF1p). HCV core and L1ORF1p interact in ribonucleoprotein complexes that traffic to lipid droplets. Despite its redistribution to HCV assembly sites, L1ORF1p is dispensable for HCV infection. In contrast, retrotransposition of engineered L1 reporter elements is restricted by HCV, correlating with an increased formation of L1ORF1p-containing cytoplasmic stress granules. Thus, our data provide first insights into the molecular interplay of endogenous transposable elements and exogenous viruses that might contribute to disease progression in vivo.
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32
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Zheng F, Li N, Xu Y, Zhou Y, Li YP. Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum. J Biol Chem 2021; 296:100018. [PMID: 33144326 PMCID: PMC7949066 DOI: 10.1074/jbc.ra120.016010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 10/22/2020] [Accepted: 11/03/2020] [Indexed: 12/14/2022] Open
Abstract
The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.
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Affiliation(s)
- Fuxiang Zheng
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Ni Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Yi Xu
- Department of Pediatric, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
| | - Yuanping Zhou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yi-Ping Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China; Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
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33
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Shimotohno K. HCV Assembly and Egress via Modifications in Host Lipid Metabolic Systems. Cold Spring Harb Perspect Med 2021; 11:cshperspect.a036814. [PMID: 32122916 PMCID: PMC7778218 DOI: 10.1101/cshperspect.a036814] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Hepatitis C virus (HCV) proliferates by hijacking the host lipid machinery. In vitro replication systems revealed many aspects of the virus life cycle; in particular, viral utilization of host lipid metabolism during HCV proliferation. HCV interacts with lipid droplets (LDs) before starting the process of virus capsid formation at the lipid-rich endoplasmic reticulum (ER) membrane compartment. HCV buds into the ER via lipoprotein assembly and secretion. Exchangeable apolipoproteins, represented by apolipoprotein E (apoE), play pivotal roles in enhancing HCV-specific infectivity. HCV virions are likely to interact with other lipoproteins circulating in blood vessels and incorporate apolipoproteins as well as lipids. This review focuses on virus assembly and egress by briefly describing the recent advances in this area.
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34
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Friend or Foe: Lipid Droplets as Organelles for Protein and Lipid Storage in Cellular Stress Response, Aging and Disease. Molecules 2020; 25:molecules25215053. [PMID: 33143278 PMCID: PMC7663626 DOI: 10.3390/molecules25215053] [Citation(s) in RCA: 45] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 10/20/2020] [Accepted: 10/22/2020] [Indexed: 02/06/2023] Open
Abstract
Lipid droplets (LDs) were considered as a mere lipid storage organelle for a long time. Recent evidence suggests that LDs are in fact distinct and dynamic organelles with a specialized proteome and functions in many cellular roles. As such, LDs contribute to cellular signaling, protein and lipid homeostasis, metabolic diseases and inflammation. In line with the multitude of functions, LDs interact with many cellular organelles including mitochondria, peroxisomes, lysosomes, the endoplasmic reticulum and the nucleus. LDs are highly mobile and dynamic organelles and impaired motility disrupts the interaction with other organelles. The reduction of interorganelle contacts results in a multitude of pathophysiologies and frequently in neurodegenerative diseases. Contacts not only supply lipids for β-oxidation in mitochondria and peroxisomes, but also may include the transfer of toxic lipids as well as misfolded and harmful proteins to LDs. Furthermore, LDs assist in the removal of protein aggregates when severe proteotoxic stress overwhelms the proteasomal system. During imbalance of cellular lipid homeostasis, LDs also support cellular detoxification. Fine-tuning of LD function is of crucial importance and many diseases are associated with dysfunctional LDs. We summarize the current understanding of LDs and their interactions with organelles, providing a storage site for harmful proteins and lipids during cellular stress, aging inflammation and various disease states.
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35
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Raut P, Glass JB, Lieberman RL. Archaeal roots of intramembrane aspartyl protease siblings signal peptide peptidase and presenilin. Proteins 2020; 89:232-241. [PMID: 32935885 DOI: 10.1002/prot.26009] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 08/27/2020] [Accepted: 09/13/2020] [Indexed: 12/21/2022]
Abstract
Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD…GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.
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Affiliation(s)
- Priyam Raut
- School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Jennifer B Glass
- School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA.,School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Raquel L Lieberman
- School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA
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36
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Alzahrani N, Wu MJ, Shanmugam S, Yi M. Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly. Viruses 2020; 12:v12101090. [PMID: 32993149 PMCID: PMC7601889 DOI: 10.3390/v12101090] [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: 07/29/2020] [Revised: 09/04/2020] [Accepted: 09/24/2020] [Indexed: 02/06/2023] Open
Abstract
The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.
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Signal Peptide Peptidase-Type Proteases: Versatile Regulators with Functions Ranging from Limited Proteolysis to Protein Degradation. J Mol Biol 2020; 432:5063-5078. [DOI: 10.1016/j.jmb.2020.05.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Revised: 05/02/2020] [Accepted: 05/19/2020] [Indexed: 12/15/2022]
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Mentrup T, Cabrera-Cabrera F, Fluhrer R, Schröder B. Physiological functions of SPP/SPPL intramembrane proteases. Cell Mol Life Sci 2020; 77:2959-2979. [PMID: 32052089 PMCID: PMC7366577 DOI: 10.1007/s00018-020-03470-6] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Revised: 01/09/2020] [Accepted: 01/22/2020] [Indexed: 01/07/2023]
Abstract
Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.
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Affiliation(s)
- Torben Mentrup
- Institute for Physiological Chemistry, Medizinisch-Theoretisches Zentrum MTZ, Technische Universität Dresden, Fiedlerstraße 42, 01307, Dresden, Germany
| | - Florencia Cabrera-Cabrera
- Institute for Physiological Chemistry, Medizinisch-Theoretisches Zentrum MTZ, Technische Universität Dresden, Fiedlerstraße 42, 01307, Dresden, Germany
| | - Regina Fluhrer
- Biochemistry and Molecular Biology, Faculty of Medicine, University of Augsburg, Universitätsstraße 2, 86135, Augsburg, Germany
- Biomedizinisches Centrum (BMC), Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 17, 81377, Munich, Germany
- DZNE-German Center for Neurodegenerative Diseases, Munich, Feodor-Lynen-Strasse 17, 81377, Munich, Germany
| | - Bernd Schröder
- Institute for Physiological Chemistry, Medizinisch-Theoretisches Zentrum MTZ, Technische Universität Dresden, Fiedlerstraße 42, 01307, Dresden, Germany.
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Vieyres G, Reichert I, Carpentier A, Vondran FWR, Pietschmann T. The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly. PLoS Pathog 2020; 16:e1008554. [PMID: 32542055 PMCID: PMC7316345 DOI: 10.1371/journal.ppat.1008554] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2019] [Revised: 06/25/2020] [Accepted: 04/15/2020] [Indexed: 12/12/2022] Open
Abstract
Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/β hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis.
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Affiliation(s)
- Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- * E-mail: (GV); (TP)
| | - Isabelle Reichert
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Arnaud Carpentier
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Florian W. R. Vondran
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
- ReMediES, Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
- * E-mail: (GV); (TP)
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The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection. Cells 2019; 8:cells8121555. [PMID: 31810281 PMCID: PMC6953030 DOI: 10.3390/cells8121555] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2019] [Revised: 11/28/2019] [Accepted: 11/29/2019] [Indexed: 12/22/2022] Open
Abstract
Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.
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Absence of Signal Peptide Peptidase, an Essential Herpes Simplex Virus 1 Glycoprotein K Binding Partner, Reduces Virus Infectivity In Vivo. J Virol 2019; 93:JVI.01309-19. [PMID: 31511378 DOI: 10.1128/jvi.01309-19] [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: 08/06/2019] [Accepted: 09/02/2019] [Indexed: 12/12/2022] Open
Abstract
We previously reported that herpes simplex virus (HSV) glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. Binding of gK to SPP is required for HSV-1 infectivity in vitro SPP is a member of the γ-secretase family, and mice lacking SPP are embryonic lethal. To determine how SPP affects HSV-1 infectivity in vivo, the SPP gene was deleted using a tamoxifen-inducible Cre recombinase driven by the ubiquitously expressed ROSA26 promoter. SPP mRNA was reduced by more than 93% in the cornea and trigeminal ganglia (TG) and by 99% in the liver of tamoxifen-injected mice, while SPP protein expression was reduced by 90% compared to the level in control mice. Mice lacking SPP had significantly less HSV-1 replication in the eye as well as reduced gK, UL20, ICP0, and gB transcripts in the cornea and TG compared to levels in control mice. In addition, reduced infiltration of CD45+, CD4+, CD8+, F4/80+, CD11c+, and NK1.1+ T cells was observed in the cornea and TG of SPP-inducible knockout mice compared to that in control mice. Finally, in the absence of SPP, latency was significantly reduced in SPP-inducible knockout mice compared to that in control mice. Thus, in this study we have generated SPP-inducible knockout mice and shown that the absence of SPP affects virus replication in the eye of ocularly infected mice and that this reduction is correlated with the interaction of gK and SPP. These results suggest that blocking this interaction may have therapeutic potential in treating HSV-1-associated eye disease.IMPORTANCE Glycoprotein K (gK) is an essential and highly conserved HSV-1 protein. Previously, we reported that gK binds to SPP, an endoplasmic reticulum (ER) protein, and blocking this binding reduces virus infectivity in vitro and also affects gK and UL20 subcellular localization. To evaluate the function of gK binding to SPP in vivo, we generated SPP-inducible knockout mice and observed the following in the absence of SPP: (i) that significantly less HSV-1 replication was seen in ocularly infected mice than in control mice; (ii) that expression of various HSV-1 genes and cellular infiltrates in the eye and trigeminal ganglia of infected mice was less than that in control mice; and (iii) that latency was significantly reduced in infected mice. Thus, blocking of gK binding to SPP may be a useful tool to control HSV-1-induced eye disease in patients with herpes stromal keratitis (HSK).
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Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States. J Virol 2019; 93:JVI.01028-19. [PMID: 31511391 DOI: 10.1128/jvi.01028-19] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 09/08/2019] [Indexed: 12/19/2022] Open
Abstract
The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle.IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.
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Pinter N, Hach CA, Hampel M, Rekhter D, Zienkiewicz K, Feussner I, Poehlein A, Daniel R, Finkernagel F, Heimel K. Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis. PLoS Pathog 2019; 15:e1007734. [PMID: 30998787 PMCID: PMC6490947 DOI: 10.1371/journal.ppat.1007734] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 04/30/2019] [Accepted: 03/27/2019] [Indexed: 11/18/2022] Open
Abstract
The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction. Biotrophic pathogens establish compatible interactions with their host to cause disease. A critical step in this process is the suppression of plant defense responses by secreted effector proteins. In the maize infecting fungus Ustilago maydis expression of effector encoding genes is coordinately upregulated at defined stages of pathogenic development in so-called effector waves. Efficient secretion of the multitude of effectors relies on the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum. Activation of the UPR is connected to the control of fungal proliferation through direct protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Here, we show that this interaction leads to functional modification of Cib1 and modulation of UPR gene expression to adapt the UPR for long-term activity in the plant. Within a core set of UPR regulated genes we identify the signal peptide peptidase Spp1 as a key factor for fungal virulence. We show that Spp1 requires its conserved catalytic activity to suppress the plant defense and cause disease. The virulence specific function of Spp1 does not involve pathways previously known to be associated with Spp1-like proteins or plant defense suppression, suggesting a novel role for Spp1 substrates in biotrophic interactions.
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Affiliation(s)
- Niko Pinter
- Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Christina Andrea Hach
- Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Martin Hampel
- Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Dmitrij Rekhter
- Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Krzysztof Zienkiewicz
- Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
- Service Unit for Metabolomics and Lipidomics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Ivo Feussner
- Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
- Service Unit for Metabolomics and Lipidomics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
| | - Anja Poehlein
- Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany
| | - Rolf Daniel
- Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany
| | - Florian Finkernagel
- Center for Tumor Biology and Immunology (ZTI), Institute of Molecular Biology and Tumor Research (IMT), Marburg, Germany
| | - Kai Heimel
- Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, Göttingen, Germany
- * E-mail:
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Vieyres G, Pietschmann T. HCV Pit Stop at the Lipid Droplet: Refuel Lipids and Put on a Lipoprotein Coat before Exit. Cells 2019; 8:cells8030233. [PMID: 30871009 PMCID: PMC6468556 DOI: 10.3390/cells8030233] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 02/28/2019] [Accepted: 03/04/2019] [Indexed: 02/07/2023] Open
Abstract
The replication cycle of the liver-tropic hepatitis C virus (HCV) is tightly connected to the host lipid metabolism, during the virus entry, replication, assembly and egress stages, but also while the virus circulates in the bloodstream. This interplay coins viral particle properties, governs viral cell tropism, and facilitates immune evasion. This review summarizes our knowledge of these interactions focusing on the late steps of the virus replication cycle. It builds on our understanding of the cell biology of lipid droplets and the biosynthesis of liver lipoproteins and attempts to explain how HCV hijacks these organelles and pathways to assemble its lipo-viro-particles. In particular, this review describes (i) the mechanisms of viral protein translocation to and from the lipid droplet surface and the orchestration of an interface between replication and assembly complexes, (ii) the importance of the triglyceride mobilization from the lipid droplets for HCV assembly, (iii) the interplay between HCV and the lipoprotein synthesis pathway including the role played by apolipoproteins in virion assembly, and finally (iv) the consequences of these complex virus–host interactions on the virion composition and its biophysical properties. The wealth of data accumulated in the past years on the role of the lipid metabolism in HCV assembly and its imprint on the virion properties will guide vaccine design efforts and reinforce our understanding of the hepatic lipid metabolism in health and disease.
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Affiliation(s)
- Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), 30625 Hannover, Germany.
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), 30625 Hannover, Germany.
- German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 38124 Braunschweig, Germany.
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Niemeyer J, Mentrup T, Heidasch R, Müller SA, Biswas U, Meyer R, Papadopoulou AA, Dederer V, Haug-Kröper M, Adamski V, Lüllmann-Rauch R, Bergmann M, Mayerhofer A, Saftig P, Wennemuth G, Jessberger R, Fluhrer R, Lichtenthaler SF, Lemberg MK, Schröder B. The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban. EMBO Rep 2019; 20:e46449. [PMID: 30733280 PMCID: PMC6399600 DOI: 10.15252/embr.201846449] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2018] [Revised: 12/21/2018] [Accepted: 12/21/2018] [Indexed: 11/09/2022] Open
Abstract
Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.
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Affiliation(s)
- Johannes Niemeyer
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
| | - Torben Mentrup
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
- Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany
| | - Ronny Heidasch
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany
| | - Stephan A Müller
- DZNE - German Center for Neurodegenerative Diseases, Munich, Germany
- Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany
| | - Uddipta Biswas
- Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany
| | - Rieke Meyer
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
| | - Alkmini A Papadopoulou
- Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany
| | - Verena Dederer
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany
| | - Martina Haug-Kröper
- Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany
| | - Vivian Adamski
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
| | | | - Martin Bergmann
- Institute of Veterinary Anatomy, Justus Liebig University of Gießen, Gießen, Germany
| | - Artur Mayerhofer
- Cell Biology, Anatomy III, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany
| | - Paul Saftig
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
| | - Gunther Wennemuth
- Institute of Anatomy, University Hospital, Duisburg-Essen University, Essen, Germany
| | - Rolf Jessberger
- Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany
| | - Regina Fluhrer
- DZNE - German Center for Neurodegenerative Diseases, Munich, Germany
- Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany
| | - Stefan F Lichtenthaler
- DZNE - German Center for Neurodegenerative Diseases, Munich, Germany
- Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany
- Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
| | - Marius K Lemberg
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany
| | - Bernd Schröder
- Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
- Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany
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Naing SH, Oliver RC, Weiss KL, Urban VS, Lieberman RL. Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering. Biophys J 2019; 114:602-608. [PMID: 29414706 DOI: 10.1016/j.bpj.2017.12.017] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Revised: 12/04/2017] [Accepted: 12/18/2017] [Indexed: 12/14/2022] Open
Abstract
Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.
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Affiliation(s)
- Swe-Htet Naing
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia
| | - Ryan C Oliver
- Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
| | - Kevin L Weiss
- Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
| | - Volker S Urban
- Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
| | - Raquel L Lieberman
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia.
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Wang F, Hu X, Zhou B. Structural characterization of plasmodial aminopeptidase: a combined molecular docking and QSAR-based in silico approaches. Mol Divers 2019; 23:965-984. [DOI: 10.1007/s11030-019-09921-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Accepted: 01/18/2019] [Indexed: 11/24/2022]
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Ríos-Ocampo WA, Navas MC, Faber KN, Daemen T, Moshage H. The cellular stress response in hepatitis C virus infection: A balancing act to promote viral persistence and host cell survival. Virus Res 2018; 263:1-8. [PMID: 30599163 DOI: 10.1016/j.virusres.2018.12.013] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2018] [Revised: 12/03/2018] [Accepted: 12/28/2018] [Indexed: 01/14/2023]
Abstract
Oxidative- and endoplasmic reticulum (ER)-stress are common events during hepatitis C virus (HCV) infection and both regulate cell survival and determine clinical outcome. In response to intrinsic and extrinsic cellular stress, different adaptive mechanisms have evolved in hepatocytes to restore cellular homeostasis like the anti-oxidant response, the unfolded protein response (UPR) and the integrated stress response (ISR). In this review, we focus on the cellular stress response in the context of acute and chronic HCV infection. The mechanisms of induction and modulation of oxidative- and ER-stress are reviewed and analyzed from both perspectives: viral persistence and cell survival. Besides, we delve into the activation of the eIF2α/ATF4 pathway and selective autophagy induction; pathways involved in the elimination of harmful viral proteins after oxidative stress induction. For this, the negative role of autophagy upon HCV infection or negative regulation of viral replication is analyzed. Finally, we hypothesize that the cellular stress response in hepatocytes plays a major role for HCV control thus acting as an important host-factor for virus clearance during the early stages of HCV infection.
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Affiliation(s)
- W Alfredo Ríos-Ocampo
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Department Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Grupo Gastrohepatología, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia.
| | - María-Cristina Navas
- Grupo Gastrohepatología, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Toos Daemen
- Department Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
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Sedeno-Monge V, Vallejo-Ruiz V, Sosa-Jurado F, Santos-Lopez G. Polymorphisms in the hepatitis C virus core and its association with development of hepatocellular carcinoma. J Biosci 2018; 42:509-521. [PMID: 29358564 DOI: 10.1007/s12038-017-9695-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Little is known about the mechanisms underlying hepatocellular carcinoma (HCC). Some studies have focused on the role of HCV viral proteins in hepatocyte transformation. In this work we have compiled and analysed current articles regarding the impact of polymorphisms in the HCV core gene and protein on the development of HCC. An exhaustive search for fulltext articles until November 2016 in PubMed database was performed using the MeSH keywords: 'hepatitis C', 'polymorphisms', 'core', 'hepatocellular cancer' and 'hepatocarcinogenesis'. Nineteen full-text articles published between 2000 and 2016 were considered. Different articles associate not only the HCC development with polymorphisms at residues 70 and 91 in the core protein, but more with mortality and treatment response. Also, different polymorphisms were found in core and other viral proteins related to HCC development. Eleven articles reported that HCC development is significantly associated with Gln/His70, four associated it with Leu91 and two more associated it with both markers together. Additional studies are necessary, including those in different types of populations worldwide, to validate the possibility of the usability and influence in chronically HCV-infected patients as well as to observe their interaction with other risk factors or prognosis and genetic markers of the host.
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Affiliation(s)
- Virginia Sedeno-Monge
- Departamento de Ciencias de la Salud, Universidad Popular Autonoma del Estado de Puebla, Puebla, Mexico
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Garcia EJ, Vevea JD, Pon LA. Lipid droplet autophagy during energy mobilization, lipid homeostasis and protein quality control. Front Biosci (Landmark Ed) 2018; 23:1552-1563. [PMID: 29293450 DOI: 10.2741/4660] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Lipid droplets (LDs) have well-established functions as sites for lipid storage and energy mobilization to meet the metabolic demands of cells. However, recent studies have expanded the roles of LDs to protein quality control. Lipophagy, or LD degradation by autophagy, plays a vital role not only in the mobilization of free fatty acids (FFAs) and lipid homeostasis at LDs, but also in the adaptation of cells to certain forms of stress including lipid imbalance. Recent studies have provided new mechanistic insights about the diverse types of lipophagy, in particular microlipophagy. This review summarizes key findings about the mechanisms and functions of lipophagy and highlights a novel function of LD microlipophagy as a mechanism to maintain endoplasmic reticulum (ER) proteostasis under conditions of lipid imbalance.
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Affiliation(s)
- Enrique J Garcia
- Department of Pathology and Cell Biology, Columbia University, New York, NY, 10032 USA
| | - Jason D Vevea
- HHMI and Dept. of Neuroscience, University of Wisconsin, Madison, WI, 53705 USA
| | - Liza A Pon
- Department of Pathology and Cell Biology, Columbia University, New York, NY, 10032 USA,
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