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

项目总结 HIV-1的传播率很低，大多数通过粘膜途径的新感染是由病毒准种中的单一变异引起的。最近在非人类灵长类动物中的研究发现，粘膜传播点含有受感染的树突状细胞，其先天反应有时无法阻止病毒传播的原因仍有待确定。允许病毒通过受体的粘膜屏障的选择性优势包括逃避限制因素和抑制固有的抗病毒反应的能力。各种HIV-1蛋白参与了这些逃避策略，包括病毒衣壳。进入后，衣壳结合运输到细胞核和合成病毒DNA所需的细胞因子，同时排斥限制因子和专门的固有传感器。多年来，我们研究了几种天然的感受器，最近，我们的研究集中在HIV-1依赖的胞质DNA感应器环磷酸腺苷合成酶(CGAS)的激活上。我们发现，聚谷氨酰胺结合蛋白1(PQBP1)是cGAS检测HIV-1DNA所必需的接头。PQBP1与HIV-1衣壳结合，从而将其鉴定为危险信号，然后在衣壳开始分解并暴露新生的病毒DNA时招募cGAs到衣壳。我们假设PQBP1通过“诱饵和开关”机制将cGAS转移到新出现的DNA上，导致cGAS激活和下游基因表达。我们鉴定了自然发生的和基因工程的衣壳变异体，它们在不同的水平上激活了cGAS，这表明衣壳结构是这一天然途径的关键决定因素。这个项目将解决我们知识中的一个关键差距，即PQBP1-衣壳相互作用和分子重排如何控制cGAS信号的强度(目标1和2)。CGAS途径主要负责HIV-1感染树突状细胞所引发的先天免疫反应，导致抗病毒因子、I型干扰素和促炎细胞因子的表达。矛盾的是，虽然抗病毒基因的诱导抑制了复制，但促炎细胞因子可以激活静止的CD4+T细胞，并可能促进感染。我们假设，HIV-1激活的cGAS信号的强度可以扭转平衡，从抗病毒反应到促炎反应，从而有助于成功的传播。同样的机制也可以在初始传播事件下游的其他淋巴组织中激活，并可能决定水库播种的过程。这个项目将解决我们知识中的另一个关键差距，即cGAS激活水平如何推动抗病毒和促炎反应之间的平衡，以及艾滋病毒-1衣壳在决定其大小方面的作用，无论是在体外还是在体内(目标3)。我们的发现将提供对决定病毒暴露结果的分子事件的洞察，这些相同的机制也可能有助于HIV-1的发病。此外，我们的发现将为设计适合HIV-1和其他涉及cGAS途径的病理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.