Mechanisms of CD4 downregulation by primate immunodeficiency viruses

该项目旨在阐明灵长类免疫缺陷病毒的Nef蛋白（即，HIV-1、HIV-2和SIV）下调病毒宿主细胞、T淋巴细胞和巨噬细胞中的CD4。Nef是一种27 - 34-kDa肉豆蔻酰化辅助蛋白，在感染早期以高水平产生。Nef是致病性的一个重要决定因素，正如一些长期非进展者（即，10年或更长时间内未出现艾滋病症状的感染患者）携带Nef基因失活突变的HIV-1毒株。因此，对Nef功能的了解可以为治疗干预提供新的途径。Nef在受感染的细胞中具有多种作用，其中最具特征的是下调CD4。与趋化因子受体一起，CD4作为HIV-1进入细胞的共受体。Nef下调CD4被认为可以防止重复感染并增加感染性颗粒的释放，从而解释了携带Nef的HIV-1毒株的更高毒力。 我们实验室以前的工作表明，HIV-1 Nef下调CD4需要网格蛋白和宿主细胞中的异源四聚体网格蛋白相关衔接蛋白-2（AP-2）复合物。此外，我们证明了这种要求源于Nef与AP-2的直接相互作用，涉及Nef中的双亮氨酸基序和AP-2的α和σ 2亚基。由于网格蛋白和AP-2介导从质膜的内吞作用，这些发现表明Nef通过加速其从宿主细胞表面的内化来下调CD4。在过去的一年里，我们发现Nef中的第二个二元酸基序与α-σ 2半复合物上的不同结合位点相互作用。Nef下调CD4需要与双亮氨酸和二酸基序的相互作用。 Nef诱导的内化后，CD4在溶酶体中被靶向降解，导致细胞中总CD4水平的显著降低。关于这些CD4下调的晚期步骤是如何完成的，我们知之甚少。一些信号受体的下调依赖于泛素化依赖性靶向多泡体（MVB）途径。我们发现，CD4是泛素化的所有四个赖氨酸残基存在于其胞质尾部。然而，Nef表达并不改变CD4泛素化的程度和模式。此外，在Nef的存在下，泛素化缺陷的CD4突变体以与野生型CD4类似的速率被内化和降解。尽管如此，Nef诱导的CD4下调依赖于MVB靶向机制。这些结果表明Nef靶向内化的CD4，用于在通往溶酶体的途中不依赖于泛素化地递送至MVB途径。 HIV-1 Nef对宿主细胞的另一个作用是上调与不变链（Ii）相关的主要组织相容性复合体（MHC-II）的未成熟II类分子。在与John Guatelli及其同事（UCSD）的合作中，我们发现这种上调是由Nef中的双亮氨酸信号与Ii的胞质尾部中的双亮氨酸信号之间的竞争引起的，以结合α-σ 2半复合物。感染期间Nef的过表达使AP-2上的二亮氨酸结合位点饱和，从而干扰二亮氨酸和AP-2介导的MHC-II-Ii从细胞表面的内吞作用。MHC-II-Ii在细胞表面的捕获防止MHC-II在核内体中获得病毒肽，从而干扰感染细胞的免疫监视。 与Eric Freed及其同事（NCI）合作，我们还研究了衔接蛋白在HIV-1从宿主细胞出芽中的作用。我们发现，GGA蛋白，这是与trans-Golgi网络网格蛋白外壳相关的单体衔接子，通过作用于小GTP结合蛋白Arf来调节HIV-1释放到细胞外空间。

This project is aimed at elucidating the mechanisms by which the Nef protein of primate immunodeficiency viruses (i.e., HIV-1, HIV-2 and SIV) downregulates CD4 in the viral host cells, T-lymphocytes and macrophages. Nef is a 27-34-kDa myristoylated accessory protein that is produced at high levels early during infection. Nef is an important determinant of pathogenicity, as demonstrated by the finding that some long-term non-progressors (i.e., infected patients who do not develop symptoms of AIDS for 10 years or longer) carry an HIV-1 strain with inactivating mutations of the Nef gene. An understanding of Nef function could thus provide new avenues for therapeutic intervention. Nef has many effects in the infected cells, the best characterized of which is the downregulation of CD4. Together with chemokine receptors, CD4 serves as a co-receptor for HIV-1 entry into cells. The downregulation of CD4 by Nef is thought to prevent superinfection and increase the release of infectious particles, thus explaining the higher virulence of Nef-carrying HIV-1 strains. Previous work from our laboratory demonstrated that the downregulation of CD4 by HIV-1 Nef requires clathrin and the heterotetrameric, clathrin-associated adaptor protein-2 (AP-2) complex in the host cells. In addition, we demonstrated that this requirement stemmed from a direct interaction of Nef with AP-2 involving a dileucine motif in Nef and the alpha and sigma2 subunits of AP-2. Since clathrin and AP-2 mediate endocytosis from the plasma membrane, these findings indicated that Nef downregulates CD4 by accelerating its internalization form the surface of host cells. This past year, we discovered that a second, diacidic motif in Nef interacts with a different binding site on the alpha-sigma2 hemicomplex. Interactions with both the dileucine and the diacidic motifs are required for Nef to downregulate CD4. Following Nef-induced internalization, CD4 is targeted for degradation in lysosomes, leading to a profound reduction of total CD4 levels in the cell. Little is known about how these late steps of CD4 downregulation are accomplished. The downregulation of some signaling receptors depends on ubiquitination-dependent targeting to the multivesicular body (MVB) pathway. We found that CD4 is ubiquitinated on all four lysine residues present in its cytosolic tail. However, Nef expression does not change the extent and pattern of CD4 ubiquitination. Moreover, in the presence of Nef, a CD4 mutant deficient in ubiquitination is internalized and degraded at rates similar to that of wild-type CD4. Nonetheless, Nef-induced CD4 downregulation is dependent on the MVB targeting machinery. These results indicate that Nef targets internalized CD4 for ubiquitination-independent delivery to the MVB pathway en route to lysosomes. Another effect of HIV-1 Nef on the host cells is the upregulation of immature class II molecules of the major histocompatibility complex (MHC-II) in association with the invariant chain (Ii). In collaboration with John Guatelli and colleagues (UCSD), we have found that this upregulation is caused by competition between dileucine signals in Nef and in the cytosolic tail of Ii for binding to the alpha-sigma2 hemicomplex. Overexpression of Nef during infection saturates the dileucine-binding site on AP-2, thus interfering with dileucine- and AP-2-mediated endocytosis of MHC-II-Ii from the cell surface. Trapping of MHC-II-Ii at the cell surface prevents MHC-II from acquiring viral peptides in endosomes, thus interfering with immune surveillance of infected cells. In collaboration with Eric Freed and colleagues (NCI), we have also examined the role of adaptor proteins in HIV-1 budding from the host cells. We found that the GGA proteins, which are monomeric adaptors associated with trans-Golgi network clathrin coats, modulate HIV-1 release into the extracellular space by acting on the small GTP-binding protein, Arf.