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

该实验室的研究员斯佳丽·施（Scarlett Shi）领导了这个项目。我们已经成功地产生了带有SARS-CoV-1和SARS-CoV-2刺突蛋白的基于hiv的假病毒，并产生了允许这些假病毒的细胞系。我们已经制定了一种方案，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刺突蛋白融合电位的蛋白酶）瞬时转染到HEK293T细胞中，稳定表达人IFITM1、IFITM2、IFITM3及其突变体。我们用HIV-SARS假病毒挑战了这些细胞，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强烈抑制sars - cov -1介导的进入，但仅轻微抑制SARS-CoV-2驱动的进入。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，表明使用TMPRSS2可能改变病毒进入细胞的途径。考虑到异位IFITM蛋白表达的这种可变影响，我们评估了IFITM蛋白在自然允许冠状病毒感染的细胞系（Caco-2、Calu-3、原代气道上皮细胞和鼻上皮细胞）中内源性表达如何影响假病毒感染。我们发现，sirna介导的IFITM2和IFITM3的敲低导致HIV-SARS-2感染增强（约3倍），而IFITM1不被敲低。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杂志上（Shi et al., 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).