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

项目摘要/摘要 我们的实验室最近证实，坏死性下垂是一种致病和靶向的宿主途径。 肺部甲型流感病毒(IAV)感染。在这项提案中，我们试图将这些发现扩展到SARS-CoV-2 因为我们有充分的理由相信，SARS-CoV-2和IAV一样，会激活坏死性下垂。我们已经确定了 SARS-CoV-2可能引发坏死性下垂的机制，并提出这种坏死性下垂是 严重新冠肺炎患者可见肺泡细胞死亡和炎性“细胞因子风暴”。重要的是 专门的RIPK3激酶抑制剂可以针对坏死性下垂，打开一种新的和意想不到的 新冠肺炎的治疗切入点。具体地说，我们发现了一种SARS-CoV-2非结构蛋白 包含一个功能性的Rhim基序，对于传播坏死性下垂信号是必不可少的。在所有已知的细胞类型中， 当激酶RIPK3参与Rhim-Rhim与其他含Rhim的Rhim相互作用时，坏死性下垂就开始了 蛋白质。例如，在IAV感染期间，RIPK3中的Rhim与IAV传感器蛋白中的Rhim相互作用 ZBP1可引发坏死性下垂。因此我们假设CoV-2蛋白中的Rhim允许它与 RIPK3激活坏死性下垂。事实上，我们发现SARS-CoV-2蛋白与RIPK3结合并激活 人类细胞中的坏死性下垂。对此负责的确切机制仍不清楚。我们还发现，所有的 三种致病冠状病毒(SARS-CoV、MERS-CoV和SARS-CoV-2)在该蛋白中有Rhim，而没有 人类适应株(HKU-1、CO43、NL63和229E)中有一株。最后，我们发现蝙蝠， SARS-CoV-2和其他致病冠状病毒的可能自然宿主编码RIPK3的一个变体，该变体包含 由非BAT RIPK3产生的单一氨基酸变化。这一变化显著抑制了坏死性下垂信号， 提示蝙蝠的坏死性下垂机制存在缺陷或不起作用。基于这些和其他 观察到，我们假设SARS-CoV-2和相关的致病冠状病毒激活了人类的坏死性下垂 肺上皮细胞，通过Rhim-Rhim相互作用，涉及CoV-2Rhim蛋白和RIPK3，以及 这种坏死性下垂引发并放大了严重的COVID患者的肺损伤和炎症- 19.我们进一步提出，蝙蝠体内抑制的坏死性下垂信号使它们能够藏匿病原体(TO 人类)Cov，没有明显的高炎症后果。在这份提案中，我们将研究如何 SARS-CoV激活了人类细胞的坏死性下垂，如果这种坏死性下垂是体内治疗的新机会 通过在SARS-CoV-2小鼠模型中评估FDA批准的和新的高效RIPK3抑制剂 感染。我们还开发了一种携带BAT RIPK3多态的敲入小鼠，并将测试是否 与对照组相比，SARS-CoV-2引发的肺部病理在这只小鼠中受到抑制。成功者 这些研究的完成将为我们提供对致病机制和进化生物学的开创性见解。 SARS-CoV-2发病机制中的坏死下垂信号及其对治疗的重要影响 严重的新冠肺炎。

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.