An Intrinsic Link between the Metabolic and Antiviral States of the Cell

细胞代谢状态和抗病毒状态之间的内在联系

• 批准号: 10702654
• 负责人: Alex Compton
• 金额: $ 65.75万
• 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/10702654
• 关键词: 2019-nCoV; Address; Anti-Inflammatory Agents; Autophagocytosis; Benefits and Risks; Biogenesis; CCI-779; Cell Line; Cell Survival; Cells; Cellular Metabolic Process; Clinical; Endocytosis; Endosomes; Epithelial Cells; FRAP1 gene; Gene-Modified; Goals; Growth; Hamsters; Human; Immune response; Immunosuppressive Agents; Infection; Influenza A virus; Innate Immune Response; Lead; Lentivirus Vector; Link; Lysosomes; Malignant Neoplasms; Manuscripts; Mediating; Metabolic; Metabolic Pathway; Multivesicular Body; Mus; Oncogenic; Pathway interactions; Patients; Peer Review; Pharmaceutical Preparations; Predisposition; Property; Proteins; Publications; Publishing; Regimen; SARS-CoV-2 infection; Signal Transduction; Sirolimus; TSG101 gene; Therapeutic; Tissues; Ubiquitination; Virus; Virus Diseases; Work; antiviral immunity; cell growth; cellular targeting; gene therapy; improved; in vivo; inhibitor; mTOR inhibition; member; mutant; scaffold; severe COVID-19; therapeutic gene; trafficking; transcription factor; tumorigenesis; vacuolar H+-ATPase

Since work on this project began in February 2017, we have made a number of key advancements. Our initial work using rapamycin on transformed epithelial cell lines revealed that mTOR inhibition confers a 4- to 20-fold enhancement of infection by lentiviral vectors and by Influenza A virus. Furthermore, we found that the rapamycin-dependent enhancement of infection is reversed by inhibitors of endosomal acidification (v-ATPase), revealing that the enhancement requires active degradation of cellular factors via the lysosomal pathway. Through a number of approaches, we show that mTOR inhibition by multiple drugs leads to lysosomal degradation of IFITM3 in an autophagy-independent manner. Instead, endocytic trafficking through multivesicular bodies is necessary to delivery of IFITM3 to lysosomes, as confirmed by a functional requirement of ESCRT member TSG101 and by inhibition of multivesicular body formation by the compound U18666A. By studying mutant IFITM3 constructs, we found that mTOR inhibition leads to clearance of IFITM2 and IFITM3 from endosomes in a manner that is dependent on endocytosis, ubiquitination, and lysosomal acidification. This work is the first instance to describe an interrelationship between mTOR, cell-intrinsic antiviral immunity, and virus entry into cells. These results have been published in 2018 (Shi et al., PNAS 115: E10069, 2018). More recently, we have compared the ability of rapamycin analogs (rapalogs) to downmodulate IFITM proteins and to enhance other virus infections, including SARS-CoV-2. Rapamycin is currently being investigated as a therapeutic anti-inflammatory compound to treat severe COVID-19. We found that some rapalogs downmodulate IFITM proteins and enhance SARS-CoV-2 infection, while others do not, laying the groundwork for a mechanistic understanding of the cellular pathways involved. Speficially, we found that some rapalogs promote IFITM downmodulate by activating TFEB, a transcription factor controlling lysosome biogenesis and function. TFEB is also required for the SARS-CoV-2 infection-enhancing effects of rapalogs, and together with our previous publication, we found TFEB triggers IFITM degradation and SARS-CoV-2 enhancement through microautophagy, an endosomal remodeling pathway. We also showed that rapalog administration in hamsters and mice increases susceptibility to experimental SARS-CoV-2 infection and viral disease in vivo. These results have been posted as a preprint in 2021 (Shi et al. bioRxiv) and the manuscript is now in revision following peer review. We now plan to study how rapalogs, which are already used clinically to inhibit cancer growth, influence the oncogenic functions of IFITM3. IFITM3 is commonly upregulated in a variety of cancers and may act as a scaffold for PI3K/Akt/mTOR signaling to favor cell survival and growth. Thus, this project has provided an opportunity for my lab to explore new avenues with relevance to the basic and clinical understanding of tumorigenesis.

自2017年2月开始实施该项目以来，我们已经制定了一些关键的 进步。我们在转化的上皮细胞系上使用雷帕霉素的初步工作显示， mTOR抑制可使慢病毒载体的感染增强4- 20倍 和甲型流感病毒。此外，我们发现，雷帕霉素依赖性增强 内体酸化抑制剂（v-ATP酶）可逆转感染，这表明 增强需要通过溶酶体途径主动降解细胞因子。通过 通过多种方法，我们发现多种药物对mTOR的抑制导致溶酶体 以自噬非依赖性方式降解IFITM 3。相反， 通过多泡体是将IFITM 3递送到溶酶体所必需的，如通过 ESCRT成员TSG 101的功能要求，并通过抑制多泡体 由化合物U18666 A形成。通过研究突变的IFITM 3构建体，我们发现mTOR 抑制导致IFITM 2和IFITM 3从内体中清除， 依赖于内吞作用、泛素化和溶酶体酸化。这项工作是第一次 描述mTOR、细胞固有抗病毒免疫和 病毒进入细胞。这些结果已于2018年发表（Shi等人，第115章： E10069，2018）。最近，我们比较了雷帕霉素类似物（rapalogs）与 下调IFITM蛋白并增强其他病毒感染，包括SARS-CoV-2。 目前正在研究雷帕霉素作为治疗性抗炎化合物来治疗 严重COVID-19。我们发现一些雷帕霉素类似物下调IFITM蛋白，并增强IFITM蛋白的表达。 SARS-CoV-2感染，而其他人则没有，这为一种机制奠定了基础。 了解相关的细胞通路。我们发现一些雷帕霉素类似物 通过激活控制溶酶体的转录因子TFEB来促进IFITM下调 生物起源和功能。TFEB也是SARS-CoV-2感染增强效应所必需的 的rapalogs，并与我们以前的出版物一起，我们发现TFEB触发IFITM 通过微自噬降解和SARS-CoV-2增强， 通路我们还表明，仓鼠和小鼠中的雷帕霉素给药增加 对实验性SARS-CoV-2感染和体内病毒性疾病的易感性。这些结果 已于2021年作为预印本发布（Shi et al. bioRxiv），手稿现已在 经同行审查后进行修订。我们现在计划研究已经使用的rapalogs 临床上抑制癌症生长，影响IFITM 3的致癌功能。IFITM 3是 通常在多种癌症中上调，并可作为PI 3 K/Akt/mTOR的支架 信号以促进细胞存活和生长。因此，这个项目提供了一个机会， 我的实验室探索与基础和临床理解相关的新途径， 肿瘤发生