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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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 月开始该项目以来，我们取得了许多关键进展。我们对转化的上皮细胞系使用雷帕霉素的初步研究表明，mTOR 抑制可使慢病毒载体和甲型流感病毒的感染增强 4 至 20 倍。此外，我们发现雷帕霉素依赖性感染增强可被内体酸化抑制剂（v-ATP酶）逆转，表明这种增强需要通过溶酶体途径主动降解细胞因子。通过多种方法，我们证明多种药物抑制 mTOR 会导致 IFITM3 以不依赖自噬的方式发生溶酶体降解。相反，通过多泡体的内吞运输对于将 IFITM3 递送至溶酶体是必要的，ESCRT 成员 TSG101 的功能要求和化合物 U18666A 对多泡体形成的抑制证实了这一点。通过研究突变型 IFITM3 构建体，我们发现 mTOR 抑制导致 IFITM2 和 IFITM3 从内体中清除，其方式依赖于内吞作用、泛素化和溶酶体酸化。这项工作首次描述了 mTOR、细胞内在抗病毒免疫和病毒进入细胞之间的相互关系。这些结果已于 2018 年发表（Shi et al., PNAS 115: E10069, 2018）。最近，我们比较了雷帕霉素类似物（rapalogs）下调 IFITM 蛋白和增强其他病毒感染（包括 SARS-CoV-2）的能力。目前正在研究雷帕霉素作为一种治疗性抗炎化合物来治疗严重的 COVID-19。我们发现一些rapalogs下调IFITM蛋白并增强SARS-CoV-2感染，而另一些则不然，这为从机制上理解所涉及的细胞途径奠定了基础。具体来说，我们发现一些rapalogs通过激活TFEB（一种控制溶酶体生物发生和功能的转录因子）来促进IFITM下调。 rapalogs 的 SARS-CoV-2 感染增强作用也需要 TFEB，结合我们之前的出版物，我们发现 TFEB 通过微自噬（一种内体重塑途径）触发 IFITM 降解和 SARS-CoV-2 增强。我们还表明，对仓鼠和小鼠施用 rapalog 会增加体内实验性 SARS-CoV-2 感染和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 等人，bioRxiv），目前手稿正在经过同行评审后进行修订。我们现在计划研究已在临床上用于抑制癌症生长的rapalogs如何影响IFITM3的致癌功能。 IFITM3 在多种癌症中通常上调，并且可以作为 PI3K/Akt/mTOR 信号转导的支架，有利于细胞存活和生长。因此，这个项目为我的实验室提供了一个探索与肿瘤发生的基础和临床理解相关的新途径的机会。