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Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor.
Journal of Virology ( IF 5.4 ) Pub Date : 2019-11-26 , DOI: 10.1128/jvi.01221-19
Vânia Passos 1, 2 , Thomas Zillinger 3 , Nicoletta Casartelli 4 , Amelie S Wachs 5 , Shuting Xu 1 , Angelina Malassa 1 , Katja Steppich 1 , Hildegard Schilling 3 , Sergej Franz 1 , Daniel Todt 6 , Eike Steinmann 6 , Kathrin Sutter 7 , Ulf Dittmer 7 , Jens Bohne 5 , Olivier Schwartz 4 , Winfried Barchet 3, 8 , Christine Goffinet 9, 10, 11
Affiliation  

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.

中文翻译:

内源性 SERINC5 蛋白作为抗 HIV-1 因子的表征。

当在产生病毒的细胞中表达时,细胞多通道跨膜蛋白 SERINC5 会降低 HIV-1 颗粒的传染性并被 HIV-1 Nef 抵消。由于缺乏具有足够特异性和敏感性的抗体,对 SERINC5 蛋白表达和亚细胞定位的研究仅限于异源表达的 SERINC5。我们通过 CRISPR/Cas9 辅助基因编辑生成了表达内源性 SERINC5 的 Jurkat T 细胞克隆,其带有细胞外暴露的血凝素 (HA) 表位 [Jurkat SERINC5(iHA 敲入) T 细胞]。这种修改使内源性 SERINC5 蛋白水平的量化成为可能,并证明了在脂筏中的主要定位。在不调节 mRNA 和蛋白质数量的情况下,干扰素 α (IFN-α) 处理以鲁索替尼敏感的方式增强了 SERINC5 的细胞表面水平。亲代和 SERINC5(iHA 敲入)T 细胞具有产生传染性野生型 HIV-1 的能力,但不具有产生 HIV-1 Δnef 突变体的能力。SERINC5 施加的感染性降低涉及病毒融合性的适度降低。内源性 SERINC5 蛋白与 HIV-1 Δnef 病毒粒子的关联始终可检测为 35-kDa 物种,而不是异源 SERINC5,后者表现为 51-kDa 物种。Nef 介导的功能性反作用与 SERINC5 的病毒粒子排除无关,认为存在作用于病毒相关 SERINC5 的 Nef 的其他反作用机制。在 HIV-1 感染的细胞中,在没有可检测到的稳态蛋白质水平变化的情况下,Nef 触发了 SERINC5 的内化。这些发现确立了内源性 SERINC5 表达和亚细胞定位的新特性,挑战了 HIV-1 Nef 介导的 SERINC5 拮抗的现有概念,并揭示了 IFN-α 在通过细胞表面积累调节 SERINC5 中的前所未有的作用。长期寻找的抗病毒因子被 HIV-1 辅助基因产物 Nef 抵消。在这里,我们通过 CRISPR/Cas9 技术设计了表达内源性 SERINC5 等位基因的 T 细胞系,这些等位基因标记有敲入的 HA 表位。这种遗传修饰使我们能够研究内源性 SERINC5 的基本特性,并验证 HIV-1 Nef 介导的 SERINC5 反作用的拟议机制。利用这种独特的资源,
更新日期:2019-11-01
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