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.

由于该项目的工作始于2017年2月，因此我们取得了许多关键进步。我们在转化的上皮细胞系上使用雷帕霉素的最初工作表明，MTOR抑制作用赋予了慢病毒载体和流感病毒感染的4至20倍。此外，我们发现雷帕霉素依赖性感染的增强被内体酸化抑制剂（V-ATPase）逆转，表明增强需要通过溶酶体途径进行细胞因子的主动降解。通过多种方法，我们表明多种药物的MTOR抑制作用会导致IFITM3以自噬非依赖性方式溶酶体降解。取而代之的是，通过ESCRT成员TSG101的功能需求和通过化合物U186666a抑制多囊体形成的功能需求证实，通过多个物体进行内吞运输对于将IFITM3传递到溶酶体是必要的。通过研究突变体IFITM3构建体，我们发现MTOR抑制作用会导致内体从内体中清除IFITM2和IFITM3，其方式取决于内吞作用，泛素化和溶酶体酸化。这项工作是描述MTOR，细胞中性抗病毒免疫和病毒进入细胞之间的相互关系的第一个实例。这些结果已于2018年发布（Shi等，PNAS 115：E10069，2018）。最近，我们比较了雷帕霉素类似物（Rapalogs）下调IFITM蛋白并增强包括SARS-COV-2在内的其他病毒感染的能力。雷帕霉素目前正在研究为一种治疗性抗炎化合物，以治疗严重的COVID-19。我们发现，一些Rapalog会下调IFITM蛋白并增强SARS-COV-2感染，而另一些则不会使SARS-COV-2感染，为机械理解所涉及的细胞途径的机械理解奠定了基础。从事依据，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。 TFEB也需要Rapalogs的SARS-COV-2感染增强作用，并且与先前的出版物一起，我们发现TFEB触发器IFITM降解和SARS-COV-2通过微自毒剂（一种内体重塑途径）增强。我们还表明，仓鼠和小鼠的Rapalog给药增加了体内实验性SARS-COV-2感染和病毒疾病的敏感性。这些结果已在2021年作为预印本（Shi等人的Biorxiv）发布，并且在同行评审后，手稿正在修订。现在，我们计划研究已经在临床上用于抑制癌症生长的Rapalog如何影响IFITM3的致癌功能。 IFITM3通常在各种癌症中上调，并且可能充当PI3K/AKT/MTOR信号传导的支架，以有利于细胞存活和生长。因此，该项目为我的实验室提供了一个机会，可以探索与肿瘤发生的基本和临床理解有关的新途径。