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: 10487090
• 负责人: Alex Compton
• 金额: $ 8.9万
• 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/10487090
• 关键词: 2019-nCoV; ACE2; Affect; Cell Culture Techniques; Cell Line; Cells; Complement; Coronavirus Infections; Data; Epithelial Cells; Goals; HIV; HIV Infections; Human; IFITM1 gene; Infection; Influenza A virus; Journals; Mediating; Nasal Epithelium; Ohio; Peptide Hydrolases; Play; Proteins; Protocols documentation; Publishing; Research; Role; Route; SARS coronavirus; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Severe Acute Respiratory Syndrome; Severity of illness; Small Interfering RNA; TMPRSS2 gene; Universities; Virus; Work; airway epithelium; base; human coronavirus; in vivo; knock-down; mutant; novel coronavirus; protein expression; protein function; receptor; respiratory virus

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).

该实验室的研究员 Scarlett Shi 正在领导这个项目。我们已成功生产出带有 SARS-CoV-1 和 SARS-CoV-2 刺突蛋白的基于 HIV 的假病毒，并生产出允许这些假病毒生长的细胞系。我们开发了一种瞬时转染人类 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）的方案)进入稳定表达人IFITM1、IFITM2、IFITM3及其突变体的HEK293T细胞中。我们用 HIV-SARS 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。鉴于异位 IFITM 蛋白表达的这种可变效应，我们评估了在天然允许冠状病毒感染的细胞系（Caco-2、Calu-3、初级气道上皮细胞和鼻上皮细胞）中内源表达的 IFITM 蛋白如何影响假病毒感染。我们发现 siRNA 介导的 IFITM2 和 IFITM3 敲除（而非 IFITM1）导致 HIV-SARS-2 的感染增强（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 Jacob Yount 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（Shi et al., EMBO J. 40: e106501, 2021）。