|
1
|
Wang Y. Rendezvous with Vaccinia Virus in the Post-smallpox Era: R&D Advances. Viruses 2023; 15:1742. [PMID: 37632084 PMCID: PMC10457812 DOI: 10.3390/v15081742] [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: 07/28/2023] [Revised: 08/13/2023] [Accepted: 08/14/2023] [Indexed: 08/27/2023] Open
Abstract
Smallpox was eradicated in less than 200 years after Edward Jenner's practice of cowpox variolation in 1796. The forty-three years of us living free of smallpox, beginning in 1979, never truly separated us from poxviruses. The recent outbreak of monkeypox in May 2022 might well warn us of the necessity of keeping up both the scientific research and public awareness of poxviruses. One of them in particular, the vaccinia virus (VACV), has been extensively studied as a vector given its broad host range, extraordinary thermal stability, and exceptional immunogenicity. Unceasing fundamental biological research on VACV provides us with a better understanding of its genetic elements, involvement in cellular signaling pathways, and modulation of host immune responses. This enables the rational design of safer and more efficacious next-generation vectors. To address the new technological advancement within the past decade in VACV research, this review covers the studies of viral immunomodulatory genes, modifications in commonly used vectors, novel mechanisms for rapid generation and purification of recombinant virus, and several other innovative approaches to studying its biology.
Collapse
Affiliation(s)
- Yuxiang Wang
- Vaccine Research Center, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA
| |
Collapse
|
|
2
|
SUMOylation Regulates BmNPV Replication by Moderating PKIP Intracellular Localization. Processes (Basel) 2022. [DOI: 10.3390/pr10020261] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
SUMOylation is a reversible covalent process between a small ubiquitin-like modifier (SUMO) and its target protein and has become a crucial regulator of protein functions. Here, we report that Bombyx mori nucleopolyhedrovirus (BmNPV) may take advantage of the host SUMOylation system to enhance its own replication, similar to many other viruses. Both the knockdown of BmSUMO by RNAi and chemical blocking by ginkgolic acid both impaired BmNPV replication. Using site mutation and pull-down assays, we found that lysine K70 of the protein kinase-interacting protein (PKIP), which is conserved in all Alphabaculoviruses, was modified by SUMO. Mutation of K70 in PKIP led to its translocation from the cytoplasm to the nucleus. Knockout and rescue experiments showed that the rescue of PKIP mutant virus with wild-type PKIP restored BmNPV replication to the normal level, but this was not true for the K70R mutation. Altogether, these results show that SUMOylation of PKIP plays a key role in BmNPV replication.
Collapse
|
|
3
|
Pérez P, Marín MQ, Lázaro-Frías A, Sorzano CÓS, Gómez CE, Esteban M, García-Arriaza J. Deletion of Vaccinia Virus A40R Gene Improves the Immunogenicity of the HIV-1 Vaccine Candidate MVA-B. Vaccines (Basel) 2020; 8:vaccines8010070. [PMID: 32041218 PMCID: PMC7158668 DOI: 10.3390/vaccines8010070] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2020] [Revised: 01/28/2020] [Accepted: 02/04/2020] [Indexed: 02/07/2023] Open
Abstract
Development of a safe and efficacious vaccine against the HIV/AIDS pandemic remains a major scientific goal. We previously described an HIV/AIDS vaccine based on the modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120 and Gag-Pol-Nef (GPN) of clade B (termed MVA-B), which showed moderate immunogenicity in phase I prophylactic and therapeutic clinical trials. Here, to improve the immunogenicity of MVA-B, we generated a novel recombinant virus, MVA-B ΔA40R, by deleting in the MVA-B genome the vaccinia virus (VACV) A40R gene, which encodes a protein with unknown immune function. The innate immune responses triggered by MVA-B ΔA40R in infected human macrophages, in comparison to parental MVA-B, revealed an increase in the mRNA expression levels of interferon (IFN)-β, IFN-induced genes, and chemokines. Compared to priming with DNA-B (a mixture of DNA-gp120 plus DNA-GPN) and boosting with MVA-B, mice immunized with a DNA-B/MVA-B ΔA40R regimen induced higher magnitude of adaptive and memory HIV-1-specific CD4+ and CD8+ T-cell immune responses that were highly polyfunctional, mainly directed against Env. and of an effector memory phenotype, together with enhanced levels of antibodies against HIV-1 gp120. Reintroduction of the A40R gene into the MVA-B ΔA40R genome (virus termed MVA-B ΔA40R-rev) promoted in infected cells high mRNA and protein A40 levels, with A40 protein localized in the cell membrane. MVA-B ΔA40R-rev significantly reduced mRNA levels of IFN-β and of several other innate immune-related genes in infected human macrophages. In immunized mice, MVA-B ΔA40R-rev reduced the magnitude of the HIV-1-specific CD4+ and CD8+ T cell responses compared to MVA-B ΔA40R. These results revealed an immunosuppressive role of the A40 protein, findings relevant for the optimization of poxvirus vectors as vaccines.
Collapse
Affiliation(s)
- Patricia Pérez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
| | - María Q. Marín
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
| | - Adrián Lázaro-Frías
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
| | - Carlos Óscar S. Sorzano
- Biocomputing Unit, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain;
| | - Carmen E. Gómez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
| | - Mariano Esteban
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
| | - Juan García-Arriaza
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; (P.P.); (M.Q.M.); (A.L.-F.); (C.E.G.); (M.E.)
- Correspondence: ; Tel.: +34-915-854-560
| |
Collapse
|
|
4
|
Wilson VG. Viral Interplay with the Host Sumoylation System. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 963:359-388. [PMID: 28197923 PMCID: PMC7121812 DOI: 10.1007/978-3-319-50044-7_21] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Viruses have evolved elaborate means to regulate diverse cellular pathways in order to create a cellular environment that facilitates viral survival and reproduction. This includes enhancing viral macromolecular synthesis and assembly, as well as preventing antiviral responses, including intrinsic, innate, and adaptive immunity. There are numerous mechanisms by which viruses mediate their effects on the host cell, and this includes targeting various cellular post-translational modification systems, including sumoylation. The wide-ranging impact of sumoylation on cellular processes such as transcriptional regulation, apoptosis, stress response, and cell cycle control makes it an attractive target for viral dysregulation. To date, proteins from both RNA and DNA virus families have been shown to be modified by SUMO conjugation, and this modification appears critical for viral protein function. More interestingly, members of the several viral families have been shown to modulate sumoylation, including papillomaviruses, adenoviruses, herpesviruses, orthomyxoviruses, filoviruses, and picornaviruses. This chapter will focus on mechanisms by which sumoylation both impacts human viruses and is used by viruses to promote viral infection and disease.
Collapse
Affiliation(s)
- Van G Wilson
- Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, 8447 HWY 47, Bryan, TX, 77807-1359, USA.
| |
Collapse
|
|
5
|
Jüdes A, Bruch A, Klassen R, Helm M, Schaffrath R. Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast. MICROBIAL CELL (GRAZ, AUSTRIA) 2016; 3:554-564. [PMID: 28357324 PMCID: PMC5349211 DOI: 10.15698/mic2016.11.539] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Accepted: 09/19/2016] [Indexed: 01/03/2023]
Abstract
Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppressed either at elevated cultivation temperatures or under sulfur starvation. In line with this, mutants with sulfur transfer defects that are linked to enzymes (Tum1, Uba4) required for Urm1 activation by thiocarboxylation (Urm1-COSH) were found to maintain drastically reduced levels of Ahp1 urmylation and mcm5s2U34 modification. Moreover, as revealed by site specific mutagenesis, the S-transfer rhodanese domain (RHD) in the E1-like activator (Uba4) crucial for Urm1-COSH formation is critical but not essential for protein urmylation and tRNA thiolation. In sum, sulfur supply, transfer and activation chemically link protein urmylation and tRNA thiolation. These are features that distinguish the ubiquitin-like modifier system Uba4•Urm1 from canonical ubiquitin family members and will help elucidate whether, in addition to their mechanistic links, the protein and tRNA modification branches of the URM1 pathway may also relate in function to one another.
Collapse
Affiliation(s)
- André Jüdes
- Universität Kassel, Institut für Biologie, FG Mikrobiologie,
Heinrich-Plett-Str. 40, 34132 Kassel, Germany
| | - Alexander Bruch
- Universität Kassel, Institut für Biologie, FG Mikrobiologie,
Heinrich-Plett-Str. 40, 34132 Kassel, Germany
| | - Roland Klassen
- Universität Kassel, Institut für Biologie, FG Mikrobiologie,
Heinrich-Plett-Str. 40, 34132 Kassel, Germany
| | - Mark Helm
- Johannes Gutenberg Universität Mainz, Institut für Pharmazie und
Biochemie, Staudinger Weg 5, 55128 Mainz, Germany
| | - Raffael Schaffrath
- Universität Kassel, Institut für Biologie, FG Mikrobiologie,
Heinrich-Plett-Str. 40, 34132 Kassel, Germany
| |
Collapse
|
|
6
|
Ji W, Guo Z, Ding NZ, He CQ. Studying classical swine fever virus: Making the best of a bad virus. Virus Res 2015; 197:35-47. [DOI: 10.1016/j.virusres.2014.12.006] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Revised: 12/02/2014] [Accepted: 12/04/2014] [Indexed: 01/04/2023]
|
|
7
|
de la Cruz-Herrera CF, Campagna M, García MA, Marcos-Villar L, Lang V, Baz-Martínez M, Gutiérrez S, Vidal A, Rodríguez MS, Esteban M, Rivas C. Activation of the double-stranded RNA-dependent protein kinase PKR by small ubiquitin-like modifier (SUMO). J Biol Chem 2014; 289:26357-26367. [PMID: 25074923 PMCID: PMC4176227 DOI: 10.1074/jbc.m114.560961] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Revised: 07/11/2014] [Indexed: 01/07/2023] Open
Abstract
The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.
Collapse
Affiliation(s)
- Carlos F de la Cruz-Herrera
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, Madrid 28049
| | - Michela Campagna
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, Madrid 28049
| | - Maria A García
- Unidad de Investigación, Hospital Universitario Virgen de las Nieves, 18014 Granada
| | - Laura Marcos-Villar
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, Madrid 28049
| | - Valerie Lang
- Ubiquitylation and Cancer Molecular Biology Laboratory, Inbiomed, San Sebastian-Donostia, 20009 Gipuzkoa, Spain
| | - Maite Baz-Martínez
- Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de Compostela, Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela E15782
| | - Sylvia Gutiérrez
- Confocal Service of Centro Nacional de Biotecnología-CSIC, Darwin 3, Madrid 28049, and
| | - Anxo Vidal
- Departamento de Fisioloxía and Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de Compostela, Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela E15782, Spain
| | - Manuel S Rodríguez
- Ubiquitylation and Cancer Molecular Biology Laboratory, Inbiomed, San Sebastian-Donostia, 20009 Gipuzkoa, Spain
| | - Mariano Esteban
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, Madrid 28049
| | - Carmen Rivas
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, Madrid 28049,; Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de Compostela, Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela E15782,.
| |
Collapse
|
|
8
|
Mattoscio D, Segré CV, Chiocca S. Viral manipulation of cellular protein conjugation pathways: The SUMO lesson. World J Virol 2013; 2:79-90. [PMID: 24175232 PMCID: PMC3785051 DOI: 10.5501/wjv.v2.i2.79] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/23/2013] [Accepted: 02/06/2013] [Indexed: 02/05/2023] Open
Abstract
Small ubiquitin-like modifier (SUMO)ylation is a key post-translational modification mechanism that controls the function of a plethora of proteins and biological processes. Given its central regulatory role, it is not surprising that it is widely exploited by viruses. A number of viral proteins are known to modify and/or be modified by the SUMOylation system to exert their function, to create a cellular environment more favorable for virus survival and propagation, and to prevent host antiviral responses. Since the SUMO pathway is a multi-step cascade, viral proteins engage with it at many levels, to advance and favor each stage of a typical infection cycle: replication, viral assembly and immune evasion. Here we review the current knowledge on the interplay between the host SUMO system and viral lifecycle.
Collapse
|
|
9
|
Everett RD, Boutell C, Hale BG. Interplay between viruses and host sumoylation pathways. Nat Rev Microbiol 2013; 11:400-11. [PMID: 23624814 DOI: 10.1038/nrmicro3015] [Citation(s) in RCA: 138] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Post-translational modification by members of the small ubiquitin-like modifier (SUMO) family of proteins is important for the regulation of many cellular proteins and pathways. As obligate parasites, viruses must engage with the host cell throughout their replication cycles, and it is therefore unsurprising that there are many examples of interplay between viral proteins and the host sumoylation system. This article reviews recent advances in this field, summarizing information on sumoylated viral proteins, the varied ways in which viruses engage with SUMO-related pathways, and the consequences of these interactions for viral replication and engagement with innate and intrinsic immunity.
Collapse
Affiliation(s)
- Roger D Everett
- MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK.
| | | | | |
Collapse
|
|
10
|
Attenuated and replication-competent vaccinia virus strains M65 and M101 with distinct biology and immunogenicity as potential vaccine candidates against pathogens. J Virol 2013; 87:6955-74. [PMID: 23596295 DOI: 10.1128/jvi.03013-12] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.
Collapse
|
|
11
|
SUMOylation affects the interferon blocking activity of the influenza A nonstructural protein NS1 without affecting its stability or cellular localization. J Virol 2013; 87:5602-20. [PMID: 23468495 DOI: 10.1128/jvi.02063-12] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Our pioneering studies on the interplay between the small ubiquitin-like modifier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ~4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.
Collapse
|
|
12
|
Sabate R, Espargaro A, Graña-Montes R, Reverter D, Ventura S. Native Structure Protects SUMO Proteins from Aggregation into Amyloid Fibrils. Biomacromolecules 2012; 13:1916-26. [DOI: 10.1021/bm3004385] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Raimon Sabate
- Departament de Fisicoquímica,
Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII s/n, E-08028-Barcelona, Spain
| | | | | | | | | |
Collapse
|
|
13
|
Wilson VG. Sumoylation at the host-pathogen interface. Biomolecules 2012; 2:203-27. [PMID: 23795346 PMCID: PMC3685863 DOI: 10.3390/biom2020203] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2012] [Revised: 03/21/2012] [Accepted: 03/27/2012] [Indexed: 12/11/2022] Open
Abstract
Many viral proteins have been shown to be sumoylated with corresponding regulatory effects on their protein function, indicating that this host cell modification process is widely exploited by viral pathogens to control viral activity. In addition to using sumoylation to regulate their own proteins, several viral pathogens have been shown to modulate overall host sumoylation levels. Given the large number of cellular targets for SUMO addition and the breadth of critical cellular processes that are regulated via sumoylation, viral modulation of overall sumoylation presumably alters the cellular environment to ensure that it is favorable for viral reproduction and/or persistence. Like some viruses, certain bacterial plant pathogens also target the sumoylation system, usually decreasing sumoylation to disrupt host anti-pathogen responses. The recent demonstration that Listeria monocytogenes also disrupts host sumoylation, and that this is required for efficient infection, extends the plant pathogen observations to a human pathogen and suggests that pathogen modulation of host sumoylation may be more widespread than previously appreciated. This review will focus on recent aspects of how pathogens modulate the host sumoylation system and how this benefits the pathogen.
Collapse
Affiliation(s)
- Van G Wilson
- Department of Microbial & Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, 8447 HWY 47, Bryan, TX 77807-1359
| |
Collapse
|
|
14
|
Rajan S, Torres J, Thompson MS, Philipson LH. SUMO downregulates GLP-1-stimulated cAMP generation and insulin secretion. Am J Physiol Endocrinol Metab 2012; 302:E714-23. [PMID: 22234371 PMCID: PMC3311292 DOI: 10.1152/ajpendo.00486.2011] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Glucagon-like peptide-1 (GLP-1)-based incretin therapy is becoming central to the treatment of type 2 diabetes. Activation of incretin hormone receptors results in rapid elevation of cAMP followed by enhanced insulin secretion. However, the incretin effect may be significantly impaired in diabetes. The objective of this study is to investigate downregulation of GLP-1 signaling by small ubiquitin-related modifier protein (SUMO). Mouse islets exposed to high glucose showed increased expression of endogenous SUMO transcripts and its conjugating enzyme Ubc-9. Overexpression of SUMO-1 in mouse insulinoma 6 (MIN6) cells and primary mouse β-cells resulted in reduced static and real-time estimates of intracellular cAMP upon receptor stimulation with exendin-4, a GLP-1 receptor (GLP-1R) agonist. GLP1-R was covalently modified by SUMO. Overexpression of SUMO-1 attenuated cell surface trafficking of GLP-1R, which resulted in significantly reduced insulin secretion when stimulated by exendin-4. Partial knock down of SUMO-conjugating enzyme Ubc-9 resulted in enhanced exendin-4-stimulated insulin secretion in mouse islets exposed to high glucose. Thus, SUMO modification of the GLP-1R could be a contributing factor to reduced incretin responsiveness. Elucidating mechanisms of GLP-1R regulation by sumoylation will help improve our understanding of incretin biology and of GLP-1-based treatment of type 2 diabetes.
Collapse
|
|
15
|
González-Santamaría J, Campagna M, García MA, Marcos-Villar L, González D, Gallego P, Lopitz-Otsoa F, Guerra S, Rodríguez MS, Esteban M, Rivas C. Regulation of vaccinia virus E3 protein by small ubiquitin-like modifier proteins. J Virol 2011; 85:12890-900. [PMID: 21957283 PMCID: PMC3233166 DOI: 10.1128/jvi.05628-11] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Accepted: 09/21/2011] [Indexed: 12/19/2022] Open
Abstract
The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3.
Collapse
Affiliation(s)
- José González-Santamaría
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Michela Campagna
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - María Angel García
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
- Unidad de Investigación, Hospital Virgen de las Nieves, Azpitarte 4, Granada 18012, Spain
| | - Laura Marcos-Villar
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Dolores González
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Pedro Gallego
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Fernando Lopitz-Otsoa
- Proteomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Building 801A, 48160 Derio, Spain
| | - Susana Guerra
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
- Department of Preventive Medicine and Public Health, Universidad Autónoma, Madrid, Spain
| | - Manuel S. Rodríguez
- Proteomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Building 801A, 48160 Derio, Spain
- Department of Biochemistry, University of the Basque Country, UPV/EHU, Leioa, Bizkaia, Spain
| | - Mariano Esteban
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Carmen Rivas
- Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
| |
Collapse
|
|
16
|
Abstract
Since posttranslational modification (PTM) by the small ubiquitin-related modifiers (SUMOs) was discovered over a decade ago, a huge number of cellular proteins have been found to be reversibly modified, resulting in alteration of differential cellular pathways. Although the molecular consequences of SUMO attachment are difficult to predict, the underlying principle of SUMOylation is altering inter- and/or intramolecular interactions of the modified substrate, changing localization, stability, and/or activity. Unsurprisingly, many different pathogens have evolved to exploit the cellular SUMO modification system due to its functional flexibility and far-reaching functional downstream consequences. Although the extensive knowledge gained so far is impressive, a definitive conclusion about the role of SUMO modification during virus infection in general remains elusive and is still restricted to a few, yet promising concepts. Based on the available data, this review aims, first, to provide a detailed overview of the current state of knowledge and, second, to evaluate the currently known common principles/molecular mechanisms of how human pathogenic microbes, especially viruses and their regulatory proteins, exploit the host cell SUMO modification system.
Collapse
|
|
17
|
Wrapping membranes around plant virus infection. Curr Opin Virol 2011; 1:388-95. [PMID: 22440840 DOI: 10.1016/j.coviro.2011.09.009] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2011] [Revised: 09/25/2011] [Accepted: 09/26/2011] [Indexed: 12/22/2022]
Abstract
Positive strand RNA viruses cause membrane modifications which are microenvironments or larger intracellular compartments, also called 'viroplasms'. These compartments serve to concentrate virus and host factors needed to produce new genomes. Forming these replication sites often involves virus induced membrane synthesis, changes in fatty acid metabolism, and viral recruitment of cellular factors to subcellular domains. Interacting viral and host factors builds the physical scaffold for replication complexes. Such virus induced changes are a visible cytopathology that has been used by plant and mammalian virologists to describe virus disease. This article describes key examples of membrane modifications that are essential for plant virus replication and intercellular transport.
Collapse
|
|
18
|
Krumova P, Meulmeester E, Garrido M, Tirard M, Hsiao HH, Bossis G, Urlaub H, Zweckstetter M, Kügler S, Melchior F, Bähr M, Weishaupt JH. Sumoylation inhibits alpha-synuclein aggregation and toxicity. ACTA ACUST UNITED AC 2011; 194:49-60. [PMID: 21746851 PMCID: PMC3135405 DOI: 10.1083/jcb.201010117] [Citation(s) in RCA: 199] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo. Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson’s disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.
Collapse
Affiliation(s)
- Petranka Krumova
- Department of Neurology and 2 Department of Biochemistry I, University of Göttingen, 37073 Göttingen, Germany
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
19
|
Gladue DP, Holinka LG, Fernandez-Sainz IJ, Prarat MV, O'Donell V, Vepkhvadze N, Lu Z, Rogers K, Risatti GR, Borca MV. Effects of the interactions of classical swine fever virus Core protein with proteins of the SUMOylation pathway on virulence in swine. Virology 2010; 407:129-36. [PMID: 20800867 DOI: 10.1016/j.virol.2010.07.040] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2010] [Revised: 07/21/2010] [Accepted: 07/26/2010] [Indexed: 02/07/2023]
Abstract
Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo.
Collapse
Affiliation(s)
- D P Gladue
- Plum Island Animal Disease Center, ARS, USDA, Greenport, NY 11944, USA.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
20
|
Shchelkunov SN. Interaction of orthopoxviruses with the cellular ubiquitin-ligase system. Virus Genes 2010; 41:309-18. [PMID: 20703935 DOI: 10.1007/s11262-010-0519-y] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2010] [Accepted: 07/28/2010] [Indexed: 02/06/2023]
Abstract
Protein modification by ubiquitin or ubiquitin-like polypeptides is important for the fate and functions of the majority of proteins in the eukaryotic cell and can be involved in regulation of various biological processes, including protein metabolism (degradation), protein transport to several cellular compartments, rearrangement of cytoskeleton, and transcription of cytoprotective genes. The accumulated experimental data suggest that the ankyrin-F-box-like and BTB-kelch-like proteins of orthopoxviruses, represented by the largest viral multigene families, interact with the cellular Cullin-1- and Cullin-3-containing ubiquitin-protein ligases, respectively. In addition, orthopoxviruses code for their own RING-domain-containing ubiquitin ligase. In this review, this author discusses the differences between variola (smallpox), monkeypox, cowpox, vaccinia, and ectromelia (mousepox) viruses in the organization of ankyrin-F-box and BTB-kelch protein families and their likely functions.
Collapse
Affiliation(s)
- Sergei N Shchelkunov
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentiev Ave. 10, Novosibirsk, Russia.
| |
Collapse
|
|
21
|
Lee CD, Yan YP, Liang SM, Wang TF. Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli. J Biomed Sci 2009; 16:69. [PMID: 19671144 PMCID: PMC2736159 DOI: 10.1186/1423-0127-16-69] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2009] [Accepted: 08/11/2009] [Indexed: 11/24/2022] Open
Abstract
Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.
Collapse
Affiliation(s)
- Chien-Der Lee
- Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
| | | | | | | |
Collapse
|
|
22
|
Mukherjee S, Thomas M, Dadgar N, Lieberman AP, Iñiguez-Lluhí JA. Small ubiquitin-like modifier (SUMO) modification of the androgen receptor attenuates polyglutamine-mediated aggregation. J Biol Chem 2009; 284:21296-306. [PMID: 19497852 PMCID: PMC2755854 DOI: 10.1074/jbc.m109.011494] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2009] [Revised: 05/29/2009] [Indexed: 01/30/2023] Open
Abstract
The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases.
Collapse
Affiliation(s)
| | - Monzy Thomas
- Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632
| | - Nahid Dadgar
- Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632
| | - Andrew P. Lieberman
- Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632
| | | |
Collapse
|
|
23
|
Abstract
Eukaryotic proteins can be modified through attachment to various small molecules and proteins. One such modification is conjugation to ubiquitin and ubiquitin-like proteins (UBLs), which controls an enormous range of physiological processes. Bound UBLs mainly regulate the interactions of proteins with other macromolecules, for example binding to the proteasome or recruitment to chromatin. The various UBL systems use related enzymes to attach specific UBLs to proteins (or other molecules), and most of these attachments are transient. There is increasing evidence suggesting that such UBL-protein modification evolved from prokaryotic sulphurtransferase systems or related enzymes. Moreover, proteins similar to UBL-conjugating enzymes and UBL-deconjugating enzymes seem to have already been widespread at the time of the last common ancestor of eukaryotes, suggesting that UBL-protein conjugation did not first evolve in eukaryotes.
Collapse
Affiliation(s)
- Mark Hochstrasser
- Yale University, Department of Molecular Biophysics & Biochemistry, 266 Whitney Avenue, PO Box 208114, New Haven, Connecticut 06520, USA.
| |
Collapse
|
|
24
|
Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways. FEBS Lett 2009; 583:607-14. [PMID: 19174161 DOI: 10.1016/j.febslet.2009.01.023] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2008] [Revised: 01/15/2009] [Accepted: 01/18/2009] [Indexed: 02/06/2023]
Abstract
Post-translational polypeptide tagging by conjugation with ubiquitin and ubiquitin-like (Ub/Ubl) molecules is a potent way to alter protein functions and/or sort specific protein targets to the proteasome for degradation. Many poxviruses interfere with the host Ub/Ubl system by encoding viral proteins that can usurp this pathway. Some of these include viral proteins of the membrane-associated RING-CH (MARCH) domain, p28/Really Interesting New Gene (RING) finger, ankyrin-repeat/F-box and Broad-complex, Tramtrack and Bric-a-Brac (BTB)/Kelch subgroups of the E3 Ub ligase superfamily. Here we describe and discuss the various strategies used by poxviruses to target and subvert the host cell Ub/Ubl systems.
Collapse
|
|
25
|
Abstract
Modification of proteins by ubiquitin and SUMO (small ubiquitin-like modifiers) is a dynamic and reversible process. Similar to the ubiquitin pathway, where the action of deubiquitinating enzymes removes ubiquitin from ubiquitin-adducts, SUMO is also removed intact from its substrates by proteases belonging to the sentrin-specific proteases (SENPs) family. In addition to their isopeptidase activity, SENPs also execute another essential function as endopeptidases by removing the short C-terminal extension from immature SUMOs. The defining characteristics of SENPs are their predicted conserved molecular scaffold-defined as members of peptidase Clan CE, conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). We discuss recent progress on the human SENPs and their substrates.
Collapse
Affiliation(s)
- Marcin Drag
- Program in Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
| | | |
Collapse
|
|
26
|
Lee CD, Sun HC, Hu SM, Chiu CF, Homhuan A, Liang SM, Leng CH, Wang TF. An improved SUMO fusion protein system for effective production of native proteins. Protein Sci 2008; 17:1241-8. [PMID: 18467498 DOI: 10.1110/ps.035188.108] [Citation(s) in RCA: 86] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.
Collapse
Affiliation(s)
- Chien-Der Lee
- Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
| | | | | | | | | | | | | | | |
Collapse
|
|
27
|
Netherton C, Moffat K, Brooks E, Wileman T. A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv Virus Res 2007; 70:101-82. [PMID: 17765705 PMCID: PMC7112299 DOI: 10.1016/s0065-3527(07)70004-0] [Citation(s) in RCA: 171] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.
Collapse
Affiliation(s)
- Christopher Netherton
- Vaccinology Group, Pirbright Laboratories, Institute for Animal Health, Surrey, United Kingdom
| | | | | | | |
Collapse
|
|
28
|
Benson MD, Li QJ, Kieckhafer K, Dudek D, Whorton MR, Sunahara RK, Iñiguez-Lluhí JA, Martens JR. SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5. Proc Natl Acad Sci U S A 2007; 104:1805-10. [PMID: 17261810 PMCID: PMC1794304 DOI: 10.1073/pnas.0606702104] [Citation(s) in RCA: 121] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2006] [Indexed: 11/18/2022] Open
Abstract
The voltage-gated potassium (Kv) channel Kv1.5 mediates the I(Kur) repolarizing current in human atrial myocytes and regulates vascular tone in multiple peripheral vascular beds. Understanding the complex regulation of Kv1.5 function is of substantial interest because it represents a promising pharmacological target for the treatment of atrial fibrillation and hypoxic pulmonary hypertension. Herein we demonstrate that posttranslational modification of Kv1.5 by small ubiquitin-like modifier (SUMO) proteins modulates Kv1.5 function. We have identified two membrane-proximal and highly conserved cytoplasmic sequences in Kv1.5 that conform to established SUMO modification sites in transcription factors. We find that Kv1.5 interacts specifically with the SUMO-conjugating enzyme Ubc9 and is a target for modification by SUMO-1, -2, and -3 in vivo. In addition, purified recombinant Kv1.5 serves as a substrate in a minimal in vitro reconstituted SUMOylation reaction. The SUMO-specific proteases SENP2 and Ulp1 efficiently deconjugate SUMO from Kv1.5 in vivo and in vitro, and disruption of the two identified target motifs results in a loss of the major SUMO-conjugated forms of Kv1.5. In whole-cell patch-clamp electrophysiological studies, loss of Kv1.5 SUMOylation, by either disruption of the conjugation sites or expression of the SUMO protease SENP2, leads to a selective approximately 15-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation. Reversible control of voltage-sensitive channels through SUMOylation constitutes a unique and likely widespread mechanism for adaptive tuning of the electrical excitability of cells.
Collapse
Affiliation(s)
- Mark D. Benson
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Qiu-Ju Li
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Katherine Kieckhafer
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - David Dudek
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Matthew R. Whorton
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Roger K. Sunahara
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Jorge A. Iñiguez-Lluhí
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| | - Jeffrey R. Martens
- Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-0632
| |
Collapse
|
|
29
|
Ju HJ, Brown JE, Ye CM, Verchot-Lubicz J. Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking. J Virol 2007; 81:1899-911. [PMID: 17151124 PMCID: PMC1797549 DOI: 10.1128/jvi.02009-06] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2006] [Accepted: 11/26/2006] [Indexed: 11/20/2022] Open
Abstract
Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.
Collapse
Affiliation(s)
- Ho-Jong Ju
- Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK 74078, USA
| | | | | | | |
Collapse
|
|
30
|
Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 2006; 22:159-80. [PMID: 16753028 DOI: 10.1146/annurev.cellbio.22.010605.093503] [Citation(s) in RCA: 1194] [Impact Index Per Article: 62.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Following the discovery of protein modification by the small, highly conserved ubiquitin polypeptide, a number of distinct ubiquitin-like proteins (Ubls) have been found to function as protein modifiers as well. These Ubls, which include SUMO, ISG15, Nedd8, and Atg8, function as critical regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy, and cell-cycle control. A growing body of data also implicates the dysregulation of Ubl-substrate modification and mutations in the Ubl-conjugation machinery in the etiology and progression of a number of human diseases. The primary aim of this review is to summarize the latest developments in our understanding of the different Ubl-protein modification systems, including the shared and unique features of these related pathways.
Collapse
Affiliation(s)
- Oliver Kerscher
- Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
| | | | | |
Collapse
|
|
31
|
Sulea T, Lindner HA, Ménard R. Structural aspects of recently discovered viral deubiquitinating activities. Biol Chem 2006; 387:853-62. [PMID: 16913834 DOI: 10.1515/bc.2006.108] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Protein ubiquitination has been identified as a regulatory mechanism in key cellular activities, and deubiquitination is recognized as an important step in processes governed by ubiquitin and ubiquitin-like modifiers. Viruses are known to target ubiquitin and ubiquitin-like modifier pathways using various strategies, including the recruitment of host deubiquitinating enzymes. Deubiquitinating activities have recently been described for proteins from three different virus families (adenovirus, coronavirus and herpesvirus), and predicted for others. This review centers on structural-functional aspects that characterize the confirmed viral deubiquitinating enzymes, and their relationships to established families of cellular deubiquitinating enzymes.
Collapse
Affiliation(s)
- Traian Sulea
- Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada.
| | | | | |
Collapse
|
|
32
|
Boggio R, Chiocca S. Viruses and sumoylation: recent highlights. Curr Opin Microbiol 2006; 9:430-6. [PMID: 16815735 PMCID: PMC7108358 DOI: 10.1016/j.mib.2006.06.008] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2006] [Accepted: 06/20/2006] [Indexed: 12/02/2022]
Abstract
Since its discovery in 1997, SUMO (small ubiquitin-like modifier) has been implicated in a range of activities, indicating that this protein is as important in the cell as ubiquitin is. Although it can function throughout the cell, it appears to be involved more in nuclear functions. The growing list of substrates that are covalently modified by SUMO includes many viral proteins; SUMO appears to facilitate viral infection of cells, making it a possible target for antiviral therapies. It therefore is important to understand how viruses manipulate the cellular sumoylation system and how sumoylation affects viral functions.
Collapse
|
|
33
|
Abstract
Poxviruses, a family of large DNA viruses, are unique among DNA viruses, because they carry out DNA replication in the cytoplasm rather than the nucleus. This process does not occur randomly, but instead, these viruses create cytoplasmic 'mini-nuclei', distinct sites that are surrounded by membranes derived from the rough endoplasmic reticulum (ER) that support viral replication. This review summarizes how distinct steps preceding cytoplasmic DNA replication, as well as replication itself, operate in the host cell. The collective data point to an important role for both the rough ER and the microtubules and indicate that these cellular structures help to co-ordinate the virus life cycle to ensure that individual steps occur at the right time and place. In a broader sense, they emphasize how viruses have evolved sophisticated ways to use host cells to optimize their life cycles to ensure efficient production of infectious progeny.
Collapse
Affiliation(s)
- Birgit Schramm
- European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany
| | | |
Collapse
|