Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor
| Autor: | Jens Bohne, Daniel Todt, Steppich K, Sergej Franz, Angelina Malassa, Thomas Zillinger, Eike Steinmann, Xu S, Nicoletta Casartelli, Passos, Christine Goffinet, Winfried Barchet, Wachs As, Oliver Schwartz, Schilling H, Kathrin Sutter, Ulf Dittmer |
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| Přispěvatelé: | Centre for Experimental and Clinical Infection Research [Hanover] (TWINCORE), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Institute of Clinical Chemistry and Clinical Pharmacology [Bonn, Allemagne], University of Bonn, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Hannover Medical School [Hannover] (MHH), Ruhr University Bochum (RUB), University of Duisburg-Essen, German Centre for Infection Research (DZIF), Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Berlin Institute of Health (BIH), Vânia Passos and Sergej Franz are supported by the Infection Biology International Ph.D. Program of the Hannover Biomedical Research School. Vânia Passos is supported by the GABBA Ph.D. Program and the Fundação para a Ciência e Tecnologia (FCT). Thomas Zillinger and Winfried Barchet acknowledge Bonfor and DZIF funding and German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]) grants EXC1023: ImmunoSensation and CRCs 670 and 704. This work is supported by DFG funding to Christine Goffinet (Collaborative Research Centre SFB900, Microbial Persistence and its Control, project C8 and Priority Program 1923, Innate Sensing and Restriction of Retroviruses, GO2153/4 grant) and funding from the HZI and the Berlin Institute of Health (BIH) to Christine Goffinet., Universidade do Porto = University of Porto, Universität Bonn = University of Bonn, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universität Duisburg-Essen = University of Duisburg-Essen [Essen], TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
MESH: Gene Editing
MESH: CRISPR-Cas Systems T-Lymphocytes Mutant Medizin HIV Infections Endogeny Jurkat cells Epitope MESH: HIV-1 MESH: Genotype Gene Knockout Techniques SERINC5 MESH: nef Gene Products Human Immunodeficiency Virus MESH: Anti-HIV Agents Internalization media_common MESH: Gene Knockout Techniques Gene Editing 0303 health sciences human immunodeficiency virus 030302 biochemistry & molecular biology virus diseases MESH: HIV Infections MESH: Gene Expression Regulation Virus-Cell Interactions 3. Good health Cell biology interferons MESH: HEK293 Cells Host-Pathogen Interactions [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology MESH: Virion MESH: Membrane Proteins MESH: Interferon-alpha Genotype Anti-HIV Agents media_common.quotation_subject Immunology Alpha interferon Heterologous Biology Microbiology Cell Line 03 medical and health sciences Virology Nitriles Humans nef Gene Products Human Immunodeficiency Virus antiviral factor CRISPR/Cas9 030304 developmental biology Nef MESH: Humans Cell Membrane MESH: Host-Pathogen Interactions Virion Interferon-alpha Membrane Proteins Subcellular localization MESH: Cell Line HEK293 Cells Pyrimidines MESH: T-Lymphocytes Gene Expression Regulation Insect Science HIV-1 Pyrazoles CRISPR-Cas Systems MESH: Pyrazoles MESH: Cell Membrane |
| Zdroj: | Journal of Virology Journal of Virology, American Society for Microbiology, 2019, 93 (24), pp.e01221-19. ⟨10.1128/JVI.01221-19⟩ Journal of Virology, 2019, 93 (24), pp.e01221-19. ⟨10.1128/JVI.01221-19⟩ Journal of virology |
| ISSN: | 0022-538X 1098-5514 |
| DOI: | 10.1128/JVI.01221-19⟩ |
| Popis: | SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions. When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions. |
| Databáze: | OpenAIRE |
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