Necroptosis in SARS-CoV-2 pathogenesis, evolution, and therapy

项目总结/摘要 我们的实验室最近暗示坏死性凋亡是一种致病性和靶向宿主途径， 甲型流感病毒（IAV）肺部感染。在本提案中，我们寻求将这些发现扩展到SARS-CoV-2 因为我们有充分的理由相信SARS-CoV-2，像IAV一样，激活坏死性凋亡。我们已经确定 SARS-CoV-2可能触发坏死性凋亡的机制，并提出这种坏死性凋亡是 在严重的COVID-19疾病中观察到肺泡细胞死亡和炎症性“细胞因子风暴”。重要的是， 坏死性凋亡可以通过专用的RIPK 3激酶抑制剂靶向，开辟了一个新的和意想不到的 COVID-19的治疗切入点。具体来说，我们已经发现SARS-CoV-2非结构蛋白 含有一个功能性RHIM基序，是传播坏死性凋亡信号所必需的。在所有已知的细胞类型中， 当激酶RIPK 3参与RHIM-RHIM相互作用时， proteins.例如，在IAV感染期间，RIPK 3中的RHIM与IAV传感器蛋白中的RHIM相互作用 ZBP 1触发坏死性凋亡。因此，我们假设CoV-2蛋白中的RHIM允许其与 RIPK 3激活坏死性凋亡。事实上，我们发现SARS-CoV-2蛋白与RIPK 3结合， 人体细胞的坏死性凋亡确切的机制尚不清楚。我们还发现， 三种致病性CoV（SARS-CoV、MERS-CoV和SARS-CoV-2）在该蛋白质中具有RHIM，而没有一种 的人适应菌株（HKU-1、CO 43、NL 63和229 E）具有一个。最后，我们发现蝙蝠， SARS-CoV-2和其他致病性CoV的可能天然宿主，编码RIPK 3的变体， 与非蝙蝠RIPK 3相比有单个氨基酸变化。这种变化显著抑制了坏死性凋亡信号传导， 这表明蝙蝠的坏死性凋亡机制是有缺陷的或不起作用的。基于这些和其他 通过观察，我们假设SARS-CoV-2和相关致病性CoV激活了人类的坏死性凋亡， 肺上皮，通过涉及含CoV-2 RHIM蛋白和RIPK 3的RHIM-RHIM相互作用，和 这种坏死性凋亡引发并放大了在严重的COVID病例中观察到的肺损伤和炎症， 19.我们进一步提出，蝙蝠中的坏死性凋亡信号减弱，使它们能够携带致病性（ 人）没有明显的高度炎症后果的CoV。在本建议中，我们将研究如何 SARS-CoV激活了人类细胞的坏死性凋亡，如果这种坏死性凋亡是一种新的体内治疗机会， 通过在SARS-CoV-2小鼠模型中评估FDA批准的新型高效RIPK 3抑制剂， 感染我们还开发了一种携带蝙蝠RIPK 3多态性的基因敲入小鼠，并将测试 与对照组相比，SARS-CoV-2引发的肺部病理学在该小鼠中减弱。成功 这些研究的完成将为人类的遗传机制和进化生物学提供开创性的见解。 坏死性凋亡信号在SARS-CoV-2发病机制中的作用， 严重的COVID-19。

PROJECT SUMMARY/ABSTRACT Our laboratory has recently implicated necroptosis as a pathogenic and targetable host pathway during pulmonary influenza A virus (IAV) infections. In this proposal, we seek to extend these findings to SARS-CoV-2 because we have strong reason to believe that SARS-CoV-2, like IAV, activates necroptosis. We have identified a mechanism by which SARS-CoV-2 may trigger necroptosis, and propose that such necroptosis underlies the alveolar cell death and inflammatory ‘cytokine storm’ observed in severe COVID-19 disease. Importantly, necroptosis can be targeted by dedicated RIPK3 kinase inhibitors, opening up a new and unanticipated therapeutic entry-point for COVID-19. Specifically, we have discovered that a SARS-CoV-2 nonstructural protein contains a functional RHIM motif that is essential for propagating necroptosis signaling. In all known cell types, necroptosis is initiated when the kinase RIPK3 engages in RHIM-RHIM interactions with other RHIM-containing proteins. For example, during IAV infection, the RHIM in RIPK3 interacts with the RHIM in the IAV sensor protein ZBP1 to trigger necroptosis. We thus hypothesized that the RHIM in the CoV-2 protein allows it to interact with RIPK3 to activate necroptosis. Indeed, we found that the SARS-CoV-2 protein engages RIPK3 and activates necroptosis in human cells. The precise mechanism responsible remains unknown. We have also found that all three pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2) have a RHIM in this protein, whereas none of the human-adapted strains (HKU-1, CO43, NL63, and 229E) possess one. Finally, we have found that bats, the likely natural hosts of SARS-CoV-2 and other pathogenic CoVs, encode a variant of RIPK3 which contains a single amino acid change from non-bat RIPK3. This change significantly dampens necroptosis signaling, suggesting that the necroptosis machinery is defective or non-functional in bats. Based on these and other observations, we hypothesize that SARS-CoV-2 and allied pathogenic CoVs activate necroptosis in human pulmonary epithelia, via a RHIM-RHIM interaction involving the CoV-2 RHIM-containing protein and RIPK3, and that such necroptosis initiates and amplifies the lung injury and inflammation seen in severe cases of COVID- 19. We further propose that dampened necroptosis signaling in bats allows them to harbor pathogenic (to humans) CoVs without apparent hyper-inflammatory consequences. In this proposal, we will examine how SARS-CoV activates necroptosis in human cells, and if such necroptosis is a new therapeutic opportunity in vivo by evaluating FDA-approved and new, high potency RIPK3 inhibitors in a mouse model of SARS-CoV-2 infection. We have also developed a knock-in mouse harboring the bat RIPK3 polymorphism, and will test if SARS-CoV-2-initiated lung pathology is dampened in this mouse, compared to controls. The successful completion of these studies will provide pioneering insight into the mechanism and evolutionary biology of necroptosis signaling in SARS-CoV-2 pathogenesis and stand to have important ramifications for the treatment of severe COVID-19.