Excessive inflammation is the primary cause of mortality in patients with severe COVID-19, yet the underlying mechanisms remain poorly understood. Our study reveals that ACE2-dependent and -independent entries of SARS-CoV-2 in epithelial cells versus myeloid cells dictate viral replication and inflammatory responses. Mechanistically, SARS-CoV-2 NSP14 potently enhances NF-κB signalling by promoting IKK phosphorylation, while SARS-CoV-2 ORF6 exerts an opposing effect. In epithelial cells, ACE2-dependent SARS-CoV-2 entry enables viral replication, with translated ORF6 suppressing NF-κB signalling. In contrast, in myeloid cells, ACE2-independent entry blocks the translation of ORF6 and other viral structural proteins due to inefficient subgenomic RNA transcription, but NSP14 could be directly translated from genomic RNA, resulting in an abortive replication but hyperactivation of the NF-κB signalling pathway for proinflammatory cytokine production. Importantly, we identified TLR1 as a critical factor responsible for viral entry and subsequent inflammatory response through interaction with E and M proteins, which could be blocked by the small-molecule inhibitor Cu-CPT22. Collectively, our findings provide molecular insights into the mechanisms by which strong viral replication but scarce inflammatory response during the early (ACE2-dependent) infection stage, followed by low viral replication and potent inflammatory response in the late (ACE2-independent) infection stage, may contribute to COVID-19 progression.

过度炎症是重症 COVID-19 患者死亡的主要原因，但其潜在机制仍知之甚少。我们的研究表明，上皮细胞与骨髓细胞中 SARS-CoV-2 的 ACE2 依赖性和非依赖性进入决定了病毒复制和炎症反应。从机制上讲，SARS-CoV-2 NSP14 通过促进 IKK 磷酸化有效增强 NF-κB 信号转导，而 SARS-CoV-2 ORF6 则发挥相反作用。在上皮细胞中，ACE2 依赖性 SARS-CoV-2 进入可实现病毒复制，翻译的 ORF6 会抑制 NF-κB 信号传导。相比之下，在骨髓细胞中，由于亚基因组 RNA 转录效率低下，ACE2 独立的进入会阻断 ORF6 和其他病毒结构蛋白的翻译，但 NSP14 可以直接从基因组 RNA 翻译，导致复制失败，但 NF-κB 过度激活促炎细胞因子产生的信号通路。重要的是，我们确定 TLR1 是通过与 E 和 M 蛋白相互作用导致病毒进入和随后炎症反应的关键因素，而这种相互作用可以被小分子抑制剂 Cu-CPT22 阻断。总的来说，我们的研究结果提供了对早期（ACE2依赖性）感染阶段病毒复制强但炎症反应稀少，随后感染后期（ACE2独立）阶段病毒复制低和强炎症反应的机制的分子见解。可能会导致 COVID-19 的进展。