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4-20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。通过一些不同的方法，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，正如ESCRT成员TSG101的功能要求所证实的那样，通过多囊体胞内转运是将IFITM3运送到溶酶体所必需的。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。此外，包括功能上调节IFITM3水平的mTOR的复合体是mTORC2。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)。我们现在已经将结果扩展到包括对HIV-1感染的影响，这涉及到对HIV-1包膜糖蛋白介导的病毒进入的研究。我们评估了雷帕霉素和其他药物对体内作为HIV-1靶点的各种细胞类型的影响，我们发现IFITM3在这些细胞中也下调。例如，从巨噬细胞中失去IFITM3，可以更大程度地感染多种HIV-1毒株。最近，我们比较了拉帕罗格下调IFITM蛋白和增强包括SARS-CoV-2在内的病毒感染的能力。我们发现，一些Rapalog执行这些功能，而另一些则不执行，这为从机制上理解所涉及的细胞通路奠定了基础。我们还在进行一项研究，以解决抗病毒蛋白IFITM和PI3K/Akt/mTOR途径组件之间的关系。我们假设IFITM3是PI3K/Akt/mTOR信号的正调控因子，因为当mTOR被雷帕霉素抑制时，IFITM3被选择性地降解。我们的初步观察表明，某些细胞类型的内源性IFITM3是Akt充分激活所必需的。具体地说，我们发现IFITM3的敲除导致Akt丝氨酸-473位的磷酸化减少。这些发现突显了IFITM3在细胞内稳态中先前未被认识到的“管家”作用。IFITM3在多种癌症中普遍上调，这一事实加强了这一发现的意义。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 preliminary work using rapamycin on transformed epithelial cell lines has 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 distinct approaches, we show that mTOR inhibition by multiple drugs leads to lysosomal degradation of IFITM3 in an autophage-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. 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. Furthermore, the complex including mTOR that functionally modulates IFITM3 levels is mTORC2. 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). We have now extended the results to include impacts on HIV-1 infection, which involves the study of virus entry mediated by HIV-1 Env glycoprotein. We have assessed the impact of rapamycin and other drugs on various cell types that serve as HIV-1 targets in vivo and we found that IFITM3 is also downregulated in these cells. The loss of IFITM3 from macrophages, for example, allows greater degrees of infection by multiple HIV-1 strains. _____More recently, we have compared the ability of rapalogs to downmodulate IFITM proteins and to enhance virus infections, including SARS-CoV-2. We found that some rapalogs perform these functions while others do not, laying the groundwork for a mechanistic understanding of the cellular pathways involved. We are also working on a study to resolve the relationship between the antiviral proteins IFITM and components of the PI3K/Akt/mTOR pathway. We hypothesized that IFITM3 is a positive regulator of PI3K/Akt/mTOR signaling due to the fact that, when mTOR is inhibited by rapamycin, IFITM3 is selectively degraded. Our initial observations indicate that endogenous IFITM3 in some cell types is necessary for full activation of Akt. Specifically, we found that knockdown of IFITM3 resulted in decreased phosphorylation of Akt at serine-473. These findings highlight a previously unrecognized "housekeeping" role for IFITM3 in cellular homeostasis. The significance of this finding is reinforced by the fact that IFITM3 is commonly upregulated in a variety of cancers. Thus, this project has provided an opportunity for my lab to explore new avenues with relevance to the basic and clinical understanding of tumorigenesis.