Inhibiting Immune Evasion by HIV-1 Nef to Facilitate Eradication

描述（由申请人提供）：HIV-1辅助蛋白的免疫逃避可能导致宿主免疫控制感染的失败，并可能使根除方法复杂化。我们将测试的假设，抑制病毒逃避宿主免疫可以有助于治疗策略。最近的数据表明，病毒基因表达的再激活不足以导致潜伏感染HIV-1的细胞死亡。细胞死亡需要细胞毒性T淋巴细胞（CTL）的杀伤活性。因此，我们的策略侧重于HIV-1 Nef，它提供病毒逃避ctl活性。Nef阻止I类MHC到达质膜，降低细胞表面病毒抗原的浓度，抑制CTL对感染细胞的杀伤。干扰Nef的这种活性应该增强CTL的能力，并促进从宿主中消灭受感染的细胞。原则上，这种策略适用于表达低水平病毒抗原的“储存库”细胞以及潜伏病毒在药理学上被重新激活的cd阳性记忆T细胞。

DESCRIPTION (provided by applicant): Immune evasion by HIV-1 accessory proteins likely contributes to the failure of host immunity to control the infection and may complicate approaches to eradication. We will test the hypothesis that inhibiting viral evasion of host immunity can contribute to a curative treatment strategy. Recent data suggest that reactivation of viral gene expression is not sufficient to cause the death of cells latently infected with HIV-1 cell-death requires the killing-activity of cytotoxic T lymphocytes (CTL). Consequently, our strategy focuses on HIV-1 Nef, which provides viral evasion of CTL-activity. Nef prevents class I MHC from reaching the plasma membrane, decreasing the concentration of viral antigens at the cell surface and inhibiting the killing of infected cells by CTL. Interference with this activity o Nef should empower CTL and facilitate the eradication of infected cells from the host. In principle, this strategy applies to "reservoir" cells expressing low levels of viral antigens as well as to CD-positive memory T cells in which latent virus is reactivated pharmacologically. During the R21 phase of this proposal, we will validate the importance of Nef in viral resistance to eradication by showing that primary T cells in which virus is reactivated from latency display reduced surface levels of MHC-I and that this reduction is Nef-dependent and associated with resistance to CTL-mediated killing. Concurrently, we will use our recent knowledge of how Nef modulates MHC-I at the molecular and structural levels to identify small molecules capable of inhibiting this activity. We will exploit our X-ray crystallographic model of the complex formed by Nef, the cytoplasmic domain of the MHC-I α-chain, and the μ subunit of the endosomal clathrin adaptor AP1 to design prototypic peptide inhibitors and to devise a high throughput screen capable of identifying small molecule lead compounds. During the R33 phase, we will broaden our approach to drug discovery by including a novel computational method that matches semi-rigid scaffold molecules displaying amino acid R groups to the structure of the complex. We will evaluate leads from this approach as well as from our high throughput screen using cell- based secondary screens and optimize them structurally. Lastly, we will use these small molecules in in vitro, ex vivo, and in vivo settings o establish that inhibiting Nef-mediated immune evasion can facilitate viral eradication.