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).

Scarlett Shi是实验室的研究员，负责这个项目。我们已经成功地产生了携带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。我们用HIV-SARS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。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、原代气道上皮细胞和鼻上皮细胞）中内源性表达的IFITM蛋白如何影响假病毒感染。我们发现，siRNA介导的IFITM 2和IFITM 3的敲低，而不是IFITM 1，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期刊上（Shi等人，40：e106501，2021）。