Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
J Virol. 2018 May 14;92(11). doi: 10.1128/JVI.00196-18. Print 2018 Jun 1.
The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NHCl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5 early, Rab7 late, and Rab11 recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.
灵长类慢病毒辅助蛋白 Nef 通过独立的内体运输途径从细胞表面下调 CD4 和主要组织相容性复合体 I（MHC-I），从而促进病毒发病机制。此外，Nef 拮抗一种新型限制因子 SERINC5（Ser5），以增加病毒感染力。为了探索 Nef 对 Ser5 的拮抗的分子机制，我们确定了 Nef 如何影响 Ser5 的表达和细胞内运输，与 CD4 和 MHC-I 进行比较。我们证实，Nef 通过下调 CD4 下调所需的类似功能基序，将 Ser5 从人类免疫缺陷病毒 1（HIV-1）病毒粒子中排除，从而下调 Ser5 的细胞表面表达。我们发现，Nef 在稳定状态下降低 Ser5 和 CD4 的表达，这可以通过 NHCl 或巴弗洛霉素 A1 处理来挽救。使用双分子荧光互补测定法在活细胞中检测到 Nef 与 Ser5 的结合，其中 Nef 膜结合对于相互作用是必需的。此外，Nef 通过受体介导的内吞作用触发 Ser5 的快速内化，并将 Ser5 重新定位到 Rab5 早期、Rab7 晚期和 Rab11 回收性内体。AP-2、Rab5、Rab7 和 Rab11 表达水平的操纵影响 Nef 依赖的 Ser5 和 CD4 下调。此外，尽管 Nef 不促进 Ser5 多泛素化，但 Ser5 的下调依赖于泛素化途径，并且 K48-和 K63-特异性泛素连接都需要下调。最后，Nef 促进 Ser5 与 LAMP1 的共定位，这通过巴弗洛霉素 A1 处理得到增强，表明 Ser5 被靶向到溶酶体进行破坏。我们得出结论，Nef 使用类似的机制下调 Ser5 和 CD4，从而将 Ser5 分类到无回头点的降解途径中以对抗其限制。人类免疫缺陷病毒（HIV）和猴免疫缺陷病毒（SIV）表达一种称为 Nef 的辅助蛋白，以促进病毒发病机制。Nef 通过下调宿主细胞表面的 CD4 和 MHC-I 来驱动免疫逃避。最近，据报道 Nef 拮抗一种新型宿主限制因子 Ser5，以增加病毒感染力。Nef 下调细胞表面的 Ser5，从而阻止其掺入病毒颗粒，导致其抗病毒活性中断。在这里，我们报告了与 CD4 和 MHC-I 相比，Nef 介导的 Ser5 下调的机制研究。我们证明 Nef 在活细胞中直接结合 Ser5，并且 Nef-Ser5 相互作用需要 Nef 与质膜的关联。随后，Nef 通过受体介导的内吞作用将 Ser5 从质膜内化，将泛素化的 Ser5 靶向内体和溶酶体进行破坏。总之，这些结果为我们对 HIV-1 感染中 Nef-Ser5 军备竞赛的持续理解提供了新的见解。
