Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival

Abstract

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

Author Summary

KSHV is the etiologic agent of KS, the most common tumor of AIDS patients worldwide. Currently, there are no therapeutics available to directly treat latent KSHV infection. This study reveals that latent KSHV infection induces endothelial cells to become glutamine addicted, similarly to cancer cells. Extracellular glutamine is required to feed the TCA cycle through glutaminolysis, a process called anaplerosis. KSHV induces protein expression of the glutamine transporter SLC1A5 and SLC1A5 expression is required for the survival of latently infected cells. KSHV also induces the expression of the proto-oncogene Myc and its binding partner Max, as well as, the nutrient-sensing transcription factor, MondoA and its binding partner Mlx. MondoA regulates SLC1A5 and glutaminolysis during latent KSHV infection, and its expression is required for the survival of latently infected endothelial cells. These studies show that glutaminolysis and a single glutamine transporter, under the regulation of MondoA, are required for the survival of latently infected cells, providing novel druggable targets for latently infected endothelial cells. This work supports that a cancer-like metabolic signature is established by latent KSHV infection, opening the door to further therapeutic targeting specifically of KSHV latently infected cells.