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

我们已经成功地产生了基于HIV和基于VSV的假病毒，其携带SARS-CoV-1和SARS-CoV-2变体的刺突蛋白，并且产生了允许这些假病毒的细胞系。我们已经开发了一种用于将人ACE 2（SARS-CoV-1和SARS-CoV-2的受体）和TMPRSS 2（激活SARS-CoV-1和SARS-CoV-2刺突蛋白融合潜力的蛋白酶）瞬时转染到稳定表达人IFITM 1、IFITM 2、IFITM 3及其突变体的HEK 293 T细胞中的方案。然后，我们用假病毒攻击这些细胞，发现人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。IFITM 3强烈抑制SARS-CoV-1介导的进入，而它仅轻微抑制SARS-CoV-2驱动的进入。此外，如果靶细胞表达TMPRSS 2，IFITM 3的抑制作用可忽略不计。这些结果表明，利用TMPRSS 2的病毒对IFITM蛋白的敏感性降低，表明TMPRSS 2的使用可能改变病毒进入细胞的途径，并且可能是病毒逃避内在免疫屏障的手段。为了补充我们的研究，我们正在与俄亥俄州州立大学的雅各布·扬特合作，在那里可以进行具有复制能力的SARS-CoV-2的感染。我们合作工作的第一章发表在EMBO期刊上（Shi等人，EMBO J.，2021年）。有趣的是，我们发现位于内体的IFITM 3限制SARS-CoV-2感染，而位于质膜的IFITM 3促进SARS-CoV-2感染。这一发现表明，SARS-CoV-2可能会在细胞表面选择IFITM 3以获得自身利益，并为SARS-CoV-2利用细胞表面的细胞进入途径而SARS-CoV-1则没有提供另一种解释。目前，我们正在研究IFITM 3促进SARS-CoV-2在质膜融合的机制，我们正在评估最近进化出的令人关注的变体，包括Omicron，是否表现出同样的能力，利用IFITM 3为自己的利益。我们将探讨两亲性螺旋及其胆固醇结合活性如何促进IFITM 3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.