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

自该项目于2017年2月启动以来，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工作揭示了mTOR抑制赋予4至20倍的感染增强，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，雷帕霉素依赖性增强感染逆转内体酸化抑制剂（v-ATP酶），揭示了增强需要通过溶酶体途径的细胞因子的主动降解。通过许多不同的方法，我们表明，mTOR抑制多种药物导致溶酶体降解IFITM 3的自噬无关的方式。相反，通过多泡体的内吞运输是将IFITM 3递送至溶酶体所必需的，如ESCRT成员TSG 101的功能要求所证实的。通过研究突变IFITM 3构建体，我们发现mTOR抑制导致IFITM 2和IFITM 3以依赖于内吞作用、泛素化和溶酶体酸化的方式从内体清除。此外，功能性调节IFITM 3水平的包括mTOR的复合物是mT 0 RC 2。这项工作是第一次描述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等人，2018年）。我们现在已经将结果扩展到包括对HIV-1感染的影响，这涉及对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IFITM 3在这些细胞中也下调。例如，T细胞和巨噬细胞中IFITM 3的丢失使得多种HIV-1毒株的感染程度更高。_我们现在正在进行一项后续研究，以解决抗病毒蛋白IFITM与PI 3 K/Akt/mTOR通路组分之间的关系。我们假设IFITM 3是PI 3 K/Akt/mTOR信号传导的正调节因子，因为当mTOR被雷帕霉素抑制时，IFITM 3被选择性降解。我们的初步观察表明，内源性IFITM 3在某些细胞类型是必要的Akt的完全激活。具体来说，我们发现IFITM 3的敲低导致Akt在丝氨酸-473处的磷酸化减少。这些发现突出了IFITM 3在细胞稳态中的先前未被认识的“管家”作用。这一发现的重要性得到了IFITM 3通常在多种癌症中上调的事实的加强。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.