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抑制可使感染增强4- 20倍，这取决于病毒攻击的性质，特别是病毒进入细胞的途径。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。通过许多不同的方法，我们表明多种药物抑制mTOR导致IFITM3以自噬独立的方式溶酶体降解。相反，正如ESCRT成员TSG101的功能需求所证实的那样，通过多泡体的内吞运输是将IFITM3运送到溶酶体的必要条件。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。此外，包括mTOR在内的功能性调节IFITM3水平的复合体是mTORC2。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., 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HIV-1 Env糖蛋白介导的病毒进入的研究。我们已经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，T细胞和巨噬细胞中IFITM3的缺失会导致多种HIV-1毒株更大程度的感染。我们现在正在进行一项后续研究，以解决抗病毒蛋白IFITM与PI3K/Akt/mTOR通路组分之间的关系。我们假设IFITM3是PI3K/Akt/mTOR信号传导的正调节因子，因为当mTOR被雷帕霉素抑制时，IFITM3被选择性降解。我们的初步观察表明，某些细胞类型中的内源性IFITM3对于Akt的完全激活是必要的。具体来说，我们发现IFITM3的敲除导致Akt丝氨酸-473位点磷酸化降低。这些发现突出了IFITM3在细胞内稳态中以前未被认识到的“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重要性。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 preliminary work using rapamycin on transformed epithelial cell lines has revealed that mTOR inhibition confers a 4- to 20-fold enhancement of infection, depending on the nature of the virus challenge and, specifically, the route of virus entry into cells. 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., 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 T cells and macrophages, for example, allows greater degrees of infection by multiple HIV-1 strains. ___We are now working on a follow-up 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 signalling 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.