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

HIV-1的Nef和Vpu辅助蛋白- 灵长类免疫缺陷病毒通过病毒包膜糖蛋白与宿主细胞表面上的CD 4和趋化因子受体（CCR 4或CXCR 5）的组合结合来靶向辅助T细胞和巨噬细胞/单核细胞。引人注目的是，感染导致CD 4的快速和持续下调，并且在较小程度上导致趋化因子受体的下调。这些病毒共受体的下调可防止重复感染，促进病毒体释放并干扰免疫应答，导致建立稳健的感染。CD 4下调对于人类免疫缺陷病毒-1（HIV-1）的生命周期是如此重要，以至于病毒基因组中编码的两种辅助蛋白Nef和Vpu致力于这一任务。事实上，Nef和Vpu对于从感染到艾滋病的进展至关重要，这一事实最好地说明了长期非进展者的存在，这些人感染了在编码这些蛋白质的基因中携带失活突变的HIV-1菌株。因此，Nef和/或Vpu的药理学或生物学扰动具有预防HIV-1的致病作用的潜力。然而，到目前为止，这种潜力尚未实现，主要是因为Nef和Vpu没有酶活性，并且它们的作用机制还没有充分了解。 HIV-1 Nef蛋白下调CD 4的结构基础- 在以前的工作中，我们在阐明Nef下调CD 4的机制方面取得了实质性进展。我们发现Nef将表面CD 4连接到内吞和溶酶体靶向机制，导致在感染早期从宿主细胞中有效和持续地去除CD 4。我们发现Nef在CD 4内化中的作用涉及与AP-2网格蛋白衔接子的相互作用。发现Nef的C-末端环中的双亮氨酸基序和二酸基序对于与AP-2 α-σ 2半复合物上的位点的相互作用和CD 4下调是必需的，但是这些相互作用的结构细节尚不清楚。在与James Hurley（NIDDK，现在在加州大学伯克利分校）的合作中，我们解决了与AP-2的α和σ 2亚基结合的Nef的晶体结构。该结构揭示了Nef双亮氨酸基序直接与α-σ 2上含有宿主双亮氨酸信号的货物蛋白的结合位点相互作用。另一方面，Nef二酸基序不直接与AP-2相互作用，但稳定了中心环的结合能力构象。此外，该结构表明Nef核心参与与α-σ 2的直接接触，同时也作为定位中央环的支架。突变与AP-2结合和CD 4下调分析相结合，证实了晶体结构中鉴定的残基的重要性。这些分析揭示的新界面不知道被任何宿主细胞跨膜蛋白使用，因此可能是Nef特异性的。如果是这样的话，它们可能代表了一个致命弱点，可以用于开发新的抗Nef药物。 宿主细胞蛋白阿利克斯/AIP 1是Nef -1将内化的CD 4靶向多泡体途径所必需的。 在通过AP-2/网格蛋白途径诱导CD 4内化后，Nef促进内化的CD 4递送至多泡体途径（MVB），以最终在溶酶体中降解。在以前的工作中，我们发现，CD 4靶向MVB通路是独立的CD 4泛素化。在与Luis daSilva（巴西圣保罗大学）的合作中，我们最近发现这种靶向依赖于Nef与阿利克斯/AIP 1的直接相互作用，AIP 1是一种与运输所需的内体分选复合物（ESCRT）机制相关的蛋白质，有助于MVB中的货物募集和管腔内囊泡形成。我们发现Nef与阿利克斯的Bro 1和V结构域相互作用。耗尽阿利克斯或过表达的阿利克斯V结构域受损的溶酶体降解的CD 4诱导Nef。相比之下，V-结构域过表达并没有阻止细胞表面通过Nef或靶向经典的泛素化依赖性MVB途径的蛋白去除CD 4。我们还表明，Nef-Alix相互作用发生在富含内化CD 4的晚期内体中。总之，这些结果表明，阿利克斯作为一个衔接子的ESCRT依赖性，泛素非依赖性靶向CD 4的MVB途径诱导Nef。

The Nef and Vpu accessory proteins of HIV-1 - Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. Structural basis for CD4 downregulation by the Nef protein of HIV-1 - In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. We discovered that the role of Nef in CD4 internalization involves an interaction with the AP-2 clathrin adaptor. A dileucine motif and a diacidic motif in a C-terminal loop of Nef were found to be essential for interaction with a site on the AP-2 alpha-sigma2 hemicomplex and for CD4 downregulation, but the structural details of these interactions were not known. In collaboration with James Hurley (NIDDK, now at UC Berkeley), we solved the crystal structure of Nef bound to the alpha and sigma2 subunits of AP-2. The structure revealed that the Nef dileucine motif directly interacts with a binding site for host dileucine-signal containing cargo proteins on alpha-sigma2. The Nef diacidic motif, on the other hand, does not directly interact with AP-2, but stabilizes a binding-competent conformation of the central loop. In addition, the structure showed that the Nef core is involved in direct contacts with alpha-sigma2 while also serving as a scaffold to position the central loop. Mutagenesis in conjunction with AP-2 binding and CD4 downregulation analyses confirmed the importance of the residues identified in the crystal structure. The new interfaces revealed by these analyses are not known to be used by any host cell transmembrane proteins and may therefore be specific for Nef. If so, they may represent an Achilles heel that could be exploited for the development of novel anti-Nef agents. The host cell protein Alix/AIP1 is required for targeting of internalized CD4 to the multivesicular body pathway by Nef - Subsequent to induction of CD4 internalization by an AP-2/clathrin pathway, Nef promotes delivery of internalized CD4 to the multivesicular body pathway (MVB) for eventual degradation in lysosomes. In previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. In collaboration with Luis daSilva (University of Sao Paulo, Brazil) we recently found that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We showed that Nef interacts with both the Bro1 and Vdomains of Alix. Depletion of Alix or overexpression of the Alix V domain impaired lysosomal degradation of CD4 induced by Nef. In contrast, V-domain overexpression did not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical, ubiquitination-dependent MVB pathway. We also showed that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, these results indicated that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.