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

我们已经成功地培育出了携带SARS-CoV-1和SARS-CoV-2变异株刺突蛋白的基于HIV和VSV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。然后我们用假病毒攻击这些细胞，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，可能是病毒逃避内在免疫屏障的一种手段。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，在那里可以进行复制能力强的SARS-CoV-2感染。我们合作工作的第一章发表在EMBO期刊上(Ship等人，EMBO J.，2021)。有趣的是，我们发现定位于内体的IFITM3抑制SARS-CoV-2的感染，而位于质膜的IFITM3促进SARS-CoV-2的感染。这一发现表明，SARS-CoV-2可能为了自身的利益而在细胞表面共用IFITM3，并为SARS-CoV-2利用细胞表面的细胞进入途径而不是SARS-CoV-1提供了另一种解释。目前，我们正在研究IFITM3促进SARS-CoV-2质膜融合的机制，我们正在评估包括Omicron在内的最近进化的变种是否表现出同样的利用IFITM3的能力。我们将探索两亲性螺旋及其胆固醇结合活性如何促进IFITM3对SARS-CoV-2的感染。

We have successfully produced HIV-based and VSV-based pseudovirus bearing the spike protein of SARS-CoV-1 and SARS-CoV-2 variants 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 then challenged these cells with the 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 and may be a means for the virus to evade intrinsic immune barriers. To complement our studies, we are collaborating with Jacob Yount at Ohio State University, where infections with replication-competent SARS-CoV-2 can be performed. The first chapter of our collaborative work was published in the EMBO Journal (Shi et al., EMBO J., 2021). Interestingly, we found that IFITM3 localized to endosomes restricts SARS-CoV-2 infection, while IFITM3 at the plasma membrane promotes SARS-CoV-2 infection. This finding demonstrated that SARS-CoV-2 may coopt IFITM3 at the cell surface for its own benefit, and provides yet another explanation for why SARS-CoV-2 exploits a cell entry pathway at the cell surface while SARS-CoV-1 does not. Currently, we are investigating the mechanisms by which IFITM3 promotes SARS-CoV-2 fusion at the plasma membrane, and we are assessing whether the recently evolved variants of concern, including Omicron, exhibit the same capacity to use IFITM3 for its own benefit. We will explore how the amphipathic helix and its cholesterol binding activity contribute to the promotion of SARS-CoV-2 infection by IFITM3.