Unraveling the Proteome of Endothelial Cells Latently Infected with KSHV Reveals Reliance on Peroxisome Lipid Metabolism and Redox Regulation

Abstract

Viruses have evolved to modulate cell signaling transduction by reprogramming the host cell machinery to establish a long-term infection and replication. By Altering host cell signaling pathways involved in metabolism, inflammation and oxidative stress, herpesviruses can establish an environment conducive for maintenance of latent infection. In this thesis, I used systems biology technologies to identify novel cellular targets of an oncogenic virus, Kaposi’s Sarcoma Associated Herpesvirus (KSHV). KSHV is the etiological agent of Kaposi’s Sarcoma (KS). KS is an endothelial cell based tumor where more than 90% of the cells are latently infected with KSHV. Therefore, elucidating novel molecular targets that are critical in the latent state would lead to treatment specific for these infected cells. I performed a global proteomics as well as phosphoproteomics screen in mock and KSHV infected endothelial cells. Combining the proteomics results with the prediction of activated transcription factors obtained from high throughput mRNA sequencing of mock and KSHV infected cells allowed the prediction of many pathways activated by KSHV. A Steiner forest analysis utilizing a database of protein-protein interactions was employed in this step. From the set of predicted activated pathways, I validated two novel pathways that are activated during KSHV latency, peroxisome biogenesis and cytoplasmic redox homeostasis. Further, I found that metabolism of very long chain fatty acids (VLCFs) in the peroxisome is required for the survival of KSHV latently infected endothelial cells and, therefore, it is critical to KSHV pathogenesis. It has been previously shown that KSHV induces oxidative stress, and I found that KSHV activation of Thioredoxin (TXN), an antioxidant protein responsible for resolving reactive oxygen species is also necessary for the survival of latently infected cells. Therefore, the systems biology approach presented here led to the identification of two pathways critical for KSHV latency. Following background (chapter 1) and methods (chapter 2), Chapter 3 shows that KSHV modulates peroxisome biogenesis and revealing a critical role for the metabolism of very long chain fatty acids (VLCFs) in the in the course of latency. In Chapter 4, I show that oxidoreductase proteins involved in regulating redox homeostasis are required during latent infection and might be responsible for regulating MAPK kinase signaling. In conclusion, this work, I demonstrate how systems biology approach was used to identify novel critical pathways required for endothelial cells latently infected with KSHV. This furthers our understanding of KSHV pathogenesis and provides potential targets for KS clinical therapies that are specific to latently infected cells.