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

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Since work on this project began in February 2017, we have made a number of key, foundational advances. 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. 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. Through multiple approaches, we show that mTOR inhibition by rapamycin and its derivatives leads to lysosomal degradation of IFITM3 in cells and in vivo via an endolysosomal remodeling pathway known as microautophagy. This work revealed for the first time an interrelationship between mTOR, cell-intrinsic antiviral immunity, and virus entry into cells. These results have been described in three papers from our lab (Shi et al., PNAS 2018, Ozog et al. Blood 2019, and Shi et al., Journal of Clinical Investigation 2022). Recently, we performed an unbiased proteomics based approach to identify E3 ligases responsible for ubiquitinating IFITM3 and initiating IFITM3 degradation in lysosomes following rapamycin treatment. This approach has identified several hits for factors which interact with IFITM3 in a rapamycin-dependent manner. The identification of protein-protein interactions enriched by rapamycin will help identify a complete set of cellular proteins that coordinate the destruction of the innate immune protein IFITM3. As such, this work may lead to identification of drug targets that could be targeted in parallel with mTOR in order to mitigate the undesired, immunosuppressive effects of rapamycin in vivo. Additionally, we have characterized the oncogenic functions of IFITM3 and its ability to promote PI3K/Akt/mTOR signaling. 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. We are characterizing the impact of IFITM3 on Akt activity and signaling pathways mediated by Akt. We are now characterizing the impact of IFITM3 on this pathway by using a variety of knockdown and knockout approaches in diverse tissue types. Thus, this project has provided an opportunity for my lab to explore new basic and clinical research directions that will lead to a better understanding of how IFITM3 and interferon signaling promote tumorigenesis. Furthermore, we have identified that cyclosporines perform a similar function as rapamycin in that they downmodulate cell-intrinsic immunity by downmodulating IFITM proteins and can be leveraged for gene therapy. More specifically, we are characterizing the molecular mechanism by which a cyclosporine derivative rapidly counteracts IFITM3 by triggering its lysosomal degradation. We are now performing experiments to examine how this derivative promotes lentivirus-mediated gene delivery in human primary tissue and organoids and in vivo. A manuscript describing these findings is currently in preparation.