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

自2017年2月该项目启动以来，我们取得了一些关键进展。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的感染增强被内体酸化（v- atp酶）抑制剂逆转，表明这种增强需要通过溶酶体途径活性降解细胞因子。通过多种方法，我们发现多种药物抑制mTOR导致IFITM3的溶酶体降解以自噬独立的方式进行。相反，通过多泡体的内吞运输是将IFITM3运送到溶酶体所必需的，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。通过研究突变体IFITM3构建体，我们发现mTOR抑制导致IFITM2和IFITM3以依赖于内吞作用、泛素化和溶酶体酸化的方式从内体中清除。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治疗COVID-19重症的治疗性抗炎化合物。我们发现一些rapalog下调IFITM蛋白并增强SARS-CoV-2感染，而其他rapalog则没有，这为对所涉及的细胞途径的机制理解奠定了基础。具体来说，我们发现一些rapalog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也是rapalogs增强SARS-CoV-2感染作用所必需的，与我们之前的出版物一起，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。我们还发现，在仓鼠和小鼠中使用rapalog可增加对实验性SARS-CoV-2感染和体内病毒性疾病的易感性。这些结果发表在2022年的Journal of Clinical Investigation上（Shi et al.）。我们现在计划研究临床已经用于抑制肿瘤生长的rapalog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信号传导的支架，促进细胞存活和生长。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。

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 were published in the Journal of Clinical Investigation in 2022 (Shi et al.). 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.