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

解读 IFITM3 在 SARS-CoV-2 感染过程中的双刃剑作用

• 批准号: 10926422
• 负责人: Alex Compton
• 金额: $ 11.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926422
• 关键词: 2019-nCoV; ACE2; Binding; Cell Culture Techniques; Cell Line; Cell membrane; Cell surface; Cells; Cholesterol; Collaborations; Complement; Coronavirus Infections; Data; Endosomes; Exhibits; Goals; HIV; Human; IFITM1 gene; Immune; Infection; Influenza A virus; Journals; Mediating; Ohio; Pathway interactions; Peptide Hydrolases; Play; Proteins; Protocols documentation; Publishing; Research; Role; Route; SARS coronavirus; SARS-CoV-2 infection; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Severity of illness; TMPRSS2 gene; Transfection; Universities; Vesicular stomatitis Indiana virus; Virus; Work; human coronavirus; in vivo; mutant; novel coronavirus; permissiveness; protein function; receptor; respiratory virus; variants of concern

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.

我们已成功生产出基于 HIV 和 VSV 的假病毒，带有 SARS-CoV-1 和 SARS-CoV-2 变体的刺突蛋白，并产生了允许这些假病毒的细胞系。我们开发了一种方案，用于将人 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）瞬时转染到稳定表达人 IFITM1、IFITM2、IFITM3 及其突变体的 HEK293T 细胞中。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 介导的细胞进入，尽管程度不同。虽然 IFITM3 强烈抑制 SARS-CoV-1 介导的进入，但它仅轻微抑制 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 TMPRSS2 的使用可能会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 Jacob Yount 合作，该大学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 et al., EMBO J., 2021）。有趣的是，我们发现位于内体的 IFITM3 限制了 SARS-CoV-2 感染，而位于质膜的 IFITM3 则促进 SARS-CoV-2 感染。这一发现表明，SARS-CoV-2 可能会为了自身利益而在细胞表面利用 IFITM3，并为为什么 SARS-CoV-2 利用细胞表面的细胞进入途径而 SARS-CoV-1 不利用提供了另一种解释。目前，我们正在研究 IFITM3 促进 SARS-CoV-2 在质膜融合的机制，并且我们正在评估最近进化的受关注变体（包括 Omicron）是否表现出相同的能力，利用 IFITM3 来实现自身利益。我们将探讨两亲性螺旋及其胆固醇结合活性如何有助于 IFITM3 促进 SARS-CoV-2 感染。