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

自2017年2月该项目开工以来，我们取得了一系列关键进展。我们在转化的上皮细胞系上使用雷帕霉素的初步工作表明，抑制mTOR可以使慢病毒载体和甲型流感病毒的感染增加4到20倍。此外，我们发现雷帕霉素依赖的感染增强可以被内体酸化抑制物(v-ATPase)逆转，这表明这种增强需要通过溶酶体途径主动降解细胞因子。通过一些方法，我们证明了多种药物抑制mTOR导致IFITM3的溶酶体以不依赖于自噬的方式降解。相反，如ESCRT成员TSG101的功能要求和化合物U18666A抑制多囊泡体形成所证实的那样，通过多囊泡体的内吞转运是将IFITM3传递到溶酶体所必需的。通过研究突变的IFITM3结构，我们发现mTOR抑制导致IFITM2和IFITM3以一种依赖于内吞作用、泛素化和溶酶体酸化的方式从内吞体内清除。这项工作是第一次描述mTOR、细胞固有的抗病毒免疫和病毒进入细胞之间的相互关系。这些成果已于2018年发表(Shih et al.，PNAS 115：E10069,2018)。最近，我们比较了雷帕霉素类似物(雷帕洛格)下调IFITM蛋白和增强其他病毒感染的能力，包括SARS-CoV-2。雷帕霉素目前正在作为一种治疗严重新冠肺炎的抗炎化合物进行研究。我们发现，一些Rapalog下调IFITM蛋白并增强SARS-CoV-2感染，而另一些则不下调，为从机制上理解相关的细胞途径奠定了基础。特别是，我们发现一些Rapalog通过激活TFEB来促进IFITM下调，TFEB是一种控制溶酶体生物发生和功能的转录因子。Rapalog的SARS-CoV-2感染增强效应也需要TFEB，我们与我们之前的文章一起发现，TFEB通过微自噬触发IFITM降解和SARS-CoV-2增强，这是一种内体重塑途径。我们还表明，在仓鼠和小鼠中使用雷帕洛格会增加实验性SARS-CoV-2感染和体内病毒疾病的易感性。这些成果已于2021年作为预印本发布(Ship等人)。BioRxiv)，该手稿正在经过同行审查后进行修订。我们现在计划研究已经在临床上用于抑制癌症生长的拉帕洛格如何影响IFITM3的致癌功能。IFITM3在多种癌症中普遍上调，可能作为PI3K/Akt/mTOR信号的支架，促进细胞生存和生长。因此，这个项目为我的实验室提供了一个机会来探索与肿瘤发生的基本和临床理解相关的新途径。

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.