Universität zu Köln

Passos, Vania, Zillinger, Thomas ORCID: 0000-0002-0866-4835, Casartelli, Nicoletta, Wachs, Amelie S., Xu, Shuting, Malassa, Angelina, Steppich, Katja, Schilling, Hildegard, Franz, Sergej, Todt, Daniel ORCID: 0000-0002-3564-1014, Steinmann, Eike ORCID: 0000-0002-3654-9965, Sutter, Kathrin ORCID: 0000-0001-6397-6551, Dittmer, Ulf, Bohne, Jens, Schwartz, Olivier, Barchet, Winfried ORCID: 0000-0002-6745-6606 and Goffinet, Christine ORCID: 0000-0002-3959-004X (2019). Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor. J. Virol., 93 (24). WASHINGTON: AMER SOC MICROBIOLOGY. ISSN 1098-5514

Full text not available from this repository.

Abstract

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-alpha) 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 Delta nef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Delta nef 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 virusassociated 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-alpha 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Passos, Vania UNSPECIFIED UNSPECIFIED UNSPECIFIED Zillinger, Thomas UNSPECIFIED orcid.org/0000-0002-0866-4835 UNSPECIFIED Casartelli, Nicoletta UNSPECIFIED UNSPECIFIED UNSPECIFIED Wachs, Amelie S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Xu, Shuting UNSPECIFIED UNSPECIFIED UNSPECIFIED Malassa, Angelina UNSPECIFIED UNSPECIFIED UNSPECIFIED Steppich, Katja UNSPECIFIED UNSPECIFIED UNSPECIFIED Schilling, Hildegard UNSPECIFIED UNSPECIFIED UNSPECIFIED Franz, Sergej UNSPECIFIED UNSPECIFIED UNSPECIFIED Todt, Daniel UNSPECIFIED orcid.org/0000-0002-3564-1014 UNSPECIFIED Steinmann, Eike UNSPECIFIED orcid.org/0000-0002-3654-9965 UNSPECIFIED Sutter, Kathrin UNSPECIFIED orcid.org/0000-0001-6397-6551 UNSPECIFIED Dittmer, Ulf UNSPECIFIED UNSPECIFIED UNSPECIFIED Bohne, Jens UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwartz, Olivier UNSPECIFIED UNSPECIFIED UNSPECIFIED Barchet, Winfried UNSPECIFIED orcid.org/0000-0002-6745-6606 UNSPECIFIED Goffinet, Christine UNSPECIFIED orcid.org/0000-0002-3959-004X UNSPECIFIED
URN:	urn:nbn:de:hbz:38-126261
DOI:	10.1128/JVI.01221-19
Journal or Publication Title:	J. Virol.
Volume:	93
Number:	24
Date:	2019
Publisher:	AMER SOC MICROBIOLOGY
Place of Publication:	WASHINGTON
ISSN:	1098-5514
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language INFECTIVITY FACTOR; HIV-1 NEF; VIRUS; RESTRICTION; RETROVIRUS; ANTAGONISM; RELEASE; FUSION; GAG; VPU Multiple languages Virology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/12626