HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
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HIV-1 Nef 通过内体/溶酶体系统下调 SERINC5 来拮抗 SERINC5 限制
DOI：
10.1128/jvi.00196-18
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发表时间：
2018-06-01
影响因子：
5.4
通讯作者：
Zheng, Yong-Hui
中科院分区：
文献类型：
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作者：
Shi, Jing;Xiong, Ran;Zheng, Yong-Hui
灵长类慢病毒辅助蛋白Nef通过独立的内体转运途径下调细胞表面的CD4和主要组织相容性复合体I类(MHC-I)，促进病毒的致病。此外，Nef拮抗一种新的限制因子SERINC5(Ser5)，以增加病毒的传染性。为了探讨Nef拮抗Ser5的分子机制，我们比较了Nef与CD4和MHC-I对Ser5表达和细胞内转运的影响。我们证实，Nef通过下调其细胞表面表达，将Ser5排除在人类免疫缺陷病毒1型(HIV-1)病毒粒子之外，这是CD4下调所需的类似功能基序。我们发现，Nef在稳态水平上降低了Ser5和CD4的表达，这是由NH4Cl或巴菲罗星A1治疗所挽救的。在活细胞中用双分子荧光互补分析检测到NEF与Ser5的结合，其中相互作用需要Nef膜结合。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重新定位为早期的Rab5(+)，晚期的Rab7(+)，以及Rab11(+)循环的内小体。操纵AP-2、Rab5、Rab7和Rab11的表达水平会影响Nef依赖的Ser5和CD4的下调。此外，尽管Nef不促进Ser5的多泛素化，但Ser5的下调依赖于泛素化途径，而K48和K63特异性的泛素连接是下调所必需的。最后，Nef促进Ser5与LAMP1的共定位，而LAMP1可被巴菲霉素A1处理增强，这表明Ser5是以溶酶体为靶点进行破坏的。我们得出结论，Nef使用类似的机制下调Ser5和CD4，将Ser5分类为不返回点的降解途径，以抵消其限制。重要的是，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，以促进病毒的致病。NEF通过下调宿主细胞表面的CD4和MHC-I来驱动体内的免疫逃逸。最近有报道称，Nef可以对抗一种新的宿主限制因子Ser5，从而增加病毒的感染力。NEF下调细胞表面Ser5，从而阻止其与病毒颗粒结合，导致其抗病毒活性中断。在这里，我们报告了Nef介导的Ser5下调与CD4和MHC-I相比的机制研究。我们证明了Nef在活细胞中直接与Ser5结合，并且Nef-Ser5的相互作用需要Nef与质膜结合。随后，Nef通过受体介导的内吞作用将Ser5从质膜内化，并将泛素化的Ser5靶向内小体和溶酶体进行破坏。总而言之，这些结果为我们对Nef-Ser5在HIV-1感染方面的军备竞赛提供了新的见解。
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 NH4Cl 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.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo 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.