Deciphering the Double-Edged Role of IFITM3 during SARS-CoV-2 Infection

Scarlett Shih是该实验室的研究员，领导着这个项目。我们已经成功地培育出了携带SARS-CoV-1和SARS-CoV-2刺突蛋白的基于HIV的伪病毒，并获得了对这些伪病毒具有容许性的细胞系。我们建立了一种将人ACE2(SARS-CoV-1和SARS-CoV-2的受体)和TMPRSS2(一种激活SARS-CoV-1和SARS-CoV-2刺突蛋白融合潜力的蛋白酶)瞬时导入稳定表达人IFITM1、IFITM2、IFITM3及其突变体的HEK293T细胞的方法。我们用HIV-SARS伪病毒攻击这些细胞，发现人类IFITM蛋白抑制SARS-CoV-1和SARS-CoV-2介导的进入细胞，尽管程度不同。IFITM3对SARS-CoV-1介导的侵袭有很强的抑制作用，而对SARS-CoV-2诱导的侵袭只有轻微的抑制作用。此外，如果靶细胞表达TMPRSS2，IFITM3的抑制作用可以忽略不计。这些结果表明，利用TMPRSS2的病毒对IFITM蛋白的敏感性降低，表明TMPRSS2的使用可能改变病毒进入细胞的途径。鉴于异位IFITM蛋白表达的这种可变效应，我们评估了在自然允许冠状病毒感染的细胞系(Caco-2、CALU-3、原代呼吸道上皮细胞和鼻腔上皮细胞)中内源性表达的IFITM蛋白如何影响假病毒感染。我们发现，siRNA介导的IFITM2和IFITM3的敲除，而不是IFITM1的敲除，导致了HIV-SARS-2的感染增加(约3倍)。为了补充我们的研究，我们正在与俄亥俄州立大学的雅各布·扬特合作，他正在用野生型SARS-CoV-2挑战我们的细胞系。我们合作工作的第一章发表在EMBO期刊上(Shih等人，EMBO J.40：e106501,2021)。

Scarlett Shi, a Research Fellow in the lab, is leading this project. We have successfully produced HIV-based pseudovirus bearing the spike protein of SARS-CoV-1 and SARS-CoV-2 and produced cell lines that are permissive to these pseudoviruses. We have developed a protocol for transiently transfecting human ACE2 (the receptor for SARS-CoV-1 and SARS-CoV-2) and TMPRSS2 (a protease that activates the fusion potential of SARS-CoV-1 and SARS-CoV-2 spike proteins) into HEK293T cells stably expressing human IFITM1, IFITM2, IFITM3, and mutants thereof. We have challenged these cells with the HIV-SARS pseudoviruses and found that the human IFITM proteins inhibit both SARS-CoV-1- and SARS-CoV-2-mediated entry into cells, albeit to different extents. Whereas IFITM3 strongly inhibits SARS-CoV-1-mediated entry, it only slightly inhibits that driven by SARS-CoV-2. Furthermore, if target cells express TMPRSS2, the inhibitory effect of IFITM3 is negligible. These results suggest that viruses utilizing TMPRSS2 have decreased sensitivity to IFITM proteins, indicating that TMPRSS2 usage may alter the virus entry route into the cell. Given this variable effect of ectopic IFITM protein expression, we assessed how IFITM proteins endogenously expressed in cell lines that are naturally permissive to coronavirus infection (Caco-2, Calu-3, primary airway epithelial cells, and nasal epithelial cells) affect pseudovirus infection. We found that siRNA-mediated knockdown of IFITM2 and IFITM3, but not IFITM1, led to enhanced infection by HIV-SARS-2 (about 3-fold). To complement our studies, we are collaborating with Jacob Yount at Ohio State University, who is challenging our cell lines with wild-type SARS-CoV-2. The first chapter of our collaborative work was published in the EMBO Journal (Shi et al., EMBO J. 40: e106501, 2021).