Determinants of HIV-1 innate immune sensing and its role in shaping the lymphoid environment.

项目摘要 HIV-1的传播率很低，大多数通过粘膜途径的新感染都是由病毒准种的单一变体引发的。最近在非人类灵长类动物中的研究发现，粘膜传播病灶含有受感染的树突状细胞，但仍有待确定为什么它们的先天反应有时无法阻止病毒传播。允许病毒穿过接受者的粘膜屏障的选择性优势包括逃避限制因子和抑制先天性抗病毒应答的能力。各种HIV-1蛋白参与这些逃避策略，包括病毒衣壳。进入后，衣壳结合细胞因子，这些细胞因子是其转运到细胞核和合成病毒DNA所必需的，同时排斥限制因子和专门的先天传感器。多年来，我们已经研究了几种先天传感器，最近，我们的研究集中在HIV-1依赖性激活的环GAMP合酶（cGAS），细胞溶质DNA传感器。我们发现多聚谷氨酰胺结合蛋白1（PQBP 1）是HIV-1 DNA的cGAS传感所需的衔接子。PQBP 1与HIV-1衣壳结合，从而将其鉴定为危险信号，然后在cGAS开始分解并显示新生病毒DNA时将其招募到衣壳。我们假设PQBP 1通过“诱饵和开关”机制将cGAS转移到新出现的DNA，导致cGAS激活和下游基因表达。我们鉴定了天然存在的和基因工程改造的衣壳变体，其将cGAS激活至不同水平，表明衣壳结构是这种先天途径的关键决定因素。本项目将解决的我们知识中的一个关键空白是PQBP 1-衣壳相互作用和分子重排如何控制cGAS信号的强度（目的1和2）。cGAS途径主要负责由树突细胞的HIV-1感染触发的先天免疫应答，导致抗病毒因子和I型干扰素以及促炎细胞因子的表达。特别地，虽然抗病毒基因的诱导抑制复制，但促炎细胞因子可以激活静息的CD 4 + T细胞并可能促进感染。我们假设，HIV-1激活的cGAS信号的强度可以使抗病毒反应与促炎反应之间的平衡发生变化，从而有助于成功传播。同样的机制也可能在其他淋巴组织中被激活，在初始传播事件的下游，并可能决定水库播种的过程。本项目将解决的我们知识中的另一个关键空白是cGAS激活水平如何驱动抗病毒和促炎反应之间的平衡以及HIV- 1衣壳在体外和体内确定其大小的作用（Aim 3）。我们的研究结果将提供深入了解决定病毒暴露结果的分子事件，这些相同的机制也可能有助于HIV-1的发病机制。此外，我们的研究结果将为HIV-1和其他涉及cGAS途径的病理学设计适当的佐剂和疫苗策略提供重要指导。

PROJECT SUMMARY HIV-1 has a low transmission rate and most new infections via the mucosal route are initiated by a single variant out of a viral quasispecies. Recent studies in non-human primates found that mucosal transmission foci contain infected dendritic cells, and it remains to be determined why their innate responses sometimes fail to prevent viral spread. Selective advantages that allow a virus to cross a recipient's mucosal barrier include the ability to evade restriction factors and suppress innate antiviral responses. Various HIV-1 proteins participate in these evasion tactics, including the viral capsid. After entry, the capsid binds cellular factors necessary for its transit to the nucleus and synthesis of viral DNA, while repelling restriction factors and specialized innate sensors. Over the years, we have studied several innate sensors and, most recently, our studies have focused on HIV-1- dependent activation of cyclic GAMP synthase (cGAS), a cytosolic DNA sensor. We discovered that polyglutamine binding protein 1 (PQBP1) is an adaptor required for cGAS sensing of HIV-1 DNA. PQBP1 binds to the HIV-1 capsid, thereby authenticating it as a danger signal, then recruits cGAS to the capsid as it begins to disassemble and reveal the nascent viral DNA. We assume that PQBP1 transfers cGAS to the emerging DNA by a "bait and switch" mechanism, resulting in cGAS activation and downstream gene expression. We identified both naturally occurring and genetically engineered capsid variants that activate cGAS to different levels, suggesting that capsid structure is a key determinant of this innate pathway. A critical gap in our knowledge that will be addressed by this project is how PQBP1-capsid interactions and molecular rearrangements control the strength of the cGAS signal (Aims 1 and 2). The cGAS pathway is predominanUy responsible for innate immune responses triggered by HIV-1 infection of dendritic cells, resulting in expression of antiviral factors and type I interferons as well as proinflammatory cytokines. Paradoxically, while induction of antiviral genes inhibits replication, proinflammatory cytokines can activate resting CD4+ T cells and may promote infection. We hypothesize that the strength of the cGAS signal activated by HIV-1 can tip the balance from antiviral to proinflammatory responses, thereby contributing to successful transmission. The same mechanism may also be activated in other lymphoid tissues, downstream of the initial transmission event, and may determine the course of reservoir seeding. Another critical gap in our knowledge that will be addressed by this project is how the level of cGAS activation drives the balance between antiviral and proinflammatory responses and the role of the HIV- 1 capsid in determining its magnitude, both in vitro and in vivo (Aim 3). Our findings will provide insight into the molecular events that determine the outcome of viral exposure, and these same mechanisms could also contribute to HIV-1 pathogenesis. Additionally, our findings will provide important guidance for the design of appropriate adjuvant and vaccine strategies for HIV-1 and other pathologies that involve the cGAS pathway.