Infection and Inflammation Department, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4029, Australia.
Infection and Inflammation Department, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4029, Australia; Australian Infectious Disease Research Centre, GVN Center of Excellence, Brisbane, QLD, Australia.
Cell Rep. 2024 Nov 26;43(11):114921. doi: 10.1016/j.celrep.2024.114921. Epub 2024 Oct 31.
Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARS-CoV-2 mouse adapted 1 (SARS-CoV-2), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2 does not replicate in the Ace2 mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2 is likely an in vitro phenomenon with no apparent implications for infection in vivo.
血管紧张素转化酶 2（ACE2）是严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的主要进入受体，但体外观察到带有刺突 E484D 取代的株系存在 ACE2 非依赖性进入。在这里，我们使用携带刺突 E484D 的 SARS-CoV-2 小鼠适应株（SARS-CoV-2）进行全基因组 CRISPR-Cas9 敲除筛选，以鉴定 ACE2 非依赖性进入机制。HEK293T 细胞中的 SARS-CoV-2 感染依赖于硫酸乙酰肝素和内吞途径，跨膜溶酶体蛋白 TMEM106B 是最重要的贡献者。虽然 SARS-CoV-2 可以有效地感染人类脑类器官和 K18-hACE2 小鼠大脑，但它不能感染 C57BL/6J 或 Ifnar 小鼠大脑。这表明，主要在大脑中表达的 TMEM106B 介导的 ACE2 非依赖性进入，并没有明显增加内源性 Ace2 表达的小鼠中 SARS-CoV-2 神经侵袭的风险。重要的是，SARS-CoV-2 不会在 Ace2 小鼠的呼吸道中复制。总体而言，这表明 SARS-CoV-2 的强大 ACE2 非依赖性感染可能是一种体外现象，对体内感染没有明显影响。
