Elucidation of the Mechanisms of Host Cell Protein Downregulation by the Nef and Vpu Proteins of HIV-1

自噬蛋白ATG 9A促进HIV-1免疫活性 Nef是由灵长类免疫缺陷病毒HIV-1、HIV-2和SIV编码的一种辅助蛋白，通过参与多种宿主细胞途径参与病毒的复制、组装、出芽、感染和免疫逃避。为了更好地了解宿主细胞蛋白在Nef功能中的作用，我们进行了串联亲和纯化-质谱分析，并鉴定了70多个HIV-1 Nef相互作用蛋白，包括自噬相关9A（ATG 9A）蛋白。ATG 9A是自噬机制的跨膜组分，自噬是一种分解代谢过程，其中细胞质组分在溶酶体隔室中降解。下拉实验表明，ATG 9A不仅与HIV-1的Nef相互作用，而且与SIV的Nef相互作用。然而，HIV-1 Nef的表达对宿主细胞中ATG 9A的水平和定位以及自噬没有影响。为了研究ATG 9A在病毒复制中的可能作用，我们使用CRISPR/Cas9敲除（KO）HeLa宫颈癌和Jurkat T细胞中的ATG 9A，并分析病毒释放和感染性。我们观察到ATG 9A KO对这些细胞中野生型（WT）或Nef缺陷型HIV-1的释放没有影响。然而，相对于从WT细胞产生的病毒，从ATG 9A-KO HeLa和Jurkat细胞产生的WT病毒的感染性分别降低了4倍和8倍。这种感染性的降低与Nef与ATG 9A的相互作用无关，并且不是由于病毒包膜（Env）糖蛋白掺入病毒中的减少。通过用水泡性口炎病毒G糖蛋白对HIV-1病毒体进行假型化来挽救HIV-1感染性的丧失。从这些研究中，我们得出结论，ATG 9A以Env依赖但Nef非依赖的方式促进HIV-1感染性。ATG 9A可以通过参与去除抑制感染性的因子或掺入增强病毒颗粒感染性的因子来促进感染性。因此，ATG 9A是与HIV-1感染性有关的一种新的宿主细胞因子。

The Autophagy Protein ATG9A Promotes HIV-1 Infectivity Nef is an accessory protein encoded by the primate immunodeficiency viruses HIV-1, HIV-2 and SIV that conttirbutes to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways. To gain a better understanding of the role of host cell proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV. However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we used CRISPR/Cas9 to knock out (KO) ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A KO had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by 4-fold and 8-fold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein. From these studies, we concluded that ATG9A promotes HIV-1 infectivity in an Env-dependent but Nef-independent manner. ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. ATG9A is thus a novel host cell factor implicated in HIV-1 infectivity.