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

自噬蛋白ATG9A促进HIV-1感染 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、萌发、感染性和免疫逃避。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，鉴定了70多个与HIV-1 Nef相互作用的蛋白，其中包括自噬相关9A(ATG9A)蛋白。ATG9A是自噬机制的跨膜组件，自噬是一种分解代谢过程，在这个过程中，细胞质成分在溶酶体内降解。下拉实验表明，ATG9A不仅与HIV-1的Nef相互作用，而且与SIV的Nef相互作用。然而，HIV-1Nef的表达对ATG9A在宿主细胞中的水平和定位以及自噬没有影响。为了探讨ATG9A在病毒复制中的可能作用，我们利用CRISPR/Cas9在HeLa宫颈癌和Jurkat T细胞中敲除(KO)ATG9A，并分析了病毒的释放和感染性。我们观察到ATG9A KO对野生型(WT)或Nef缺陷的HIV-1在这些细胞中的释放没有影响。然而，ATG9A-KO HeLa和Jurkat细胞产生的WT病毒的感染力分别比WT细胞产生的病毒低4倍和8倍。这种传染性的降低与Nef与ATG9A的相互作用无关，也不是由于病毒包膜(Env)糖蛋白掺入病毒的减少所致。通过用水泡性口炎病毒G糖蛋白对HIV-1病毒粒子进行假型，挽救了HIV-1感染性的丧失。从这些研究中，我们得出结论，ATG9A以一种环境依赖但不依赖Nef的方式促进HIV-1的传染性。ATG9A可以通过参与去除抑制传染性的因素或加入增强病毒颗粒传染性的因素来促进传染性。因此，ATG9A是一种新的宿主细胞因子，与HIV-1的感染性有关。

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.