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Characterization of Endogenous SERINC5 Protein as anti-HIV-1 Factor.
Passos, Vânia Zillinger, Thomas Casartelli, Nicoletta Wachs, Amelie S Xu, Shuting Malassa, Angelina Steppich, Katja Schilling, Hildegard Franz, Sergej Todt, Daniel
Passos, Vânia
Zillinger, Thomas
Casartelli, Nicoletta
Wachs, Amelie S
Xu, Shuting
Malassa, Angelina
Steppich, Katja
Schilling, Hildegard
Franz, Sergej
Todt, Daniel
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2019-10-09
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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 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. 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 wildtype HIV-1, but not HIV-1 Δnef SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. Association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35 kDa species, as opposed to heterologous SERINC5 that presented as 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 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 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.
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J Virol. 2019 Oct 9. pii: JVI.01221-19. doi: 10.1128/JVI.01221-19
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en
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1098-5514
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Attribution-NonCommercial-ShareAlike 4.0 International