Bcl-xL is required to protect endothelial cells latently infected with KSHV from virus induced intrinsic apoptosis

Abstract

Kaposi’s Sarcoma Herpesvirus (KSHV) is an oncogenic gammaherpesvirus that, like other herpesviruses, can establish both lytic and latent replication programs. It is the etiologic agent of Kaposi’s Sarcoma (KS), Multicentric Castleman’s Disease (MCD), and Primary Effusion Lymphoma (PEL). Within Kaposi’s Sarcoma tumors, cells are predominantly latently infected. This poses a significant problem, as all current antivirals for herpesviruses target cells that are lytically infected, KSHV included. It is necessary to develop therapeutics that can target latently infected cells to effectively eradicate herpesvirus infections. Unfortunately, latent viruses are difficult to target since they do not produce infectious virions and they dramatically repress expression of viral genes. Targeting host cell requirement for latency, though, is a potentially viable course of action. Previously our lab performed a CRISPR/Cas9 essentiality screen in endothelial cells latently infected with KSHV. In this thesis work, I follow up on the most promising hit from that screen, Bcl-xL. Bcl-xL is an anti-apoptotic protein in the Bcl-2 protein family. The Bcl-2 family proteins are the master regulators of intrinsic apoptosis. I found that Bcl-xL is required for the survival of KSHV latently infected endothelial cells. Bcl-xL is needed during KSHV infection to inhibit virus induced apoptosis by sequestering the pro-apoptotic pore-former Bax. Bcl-xL, but not other anti-apoptotic Bcl-2 family proteins, is uniquely required because endothelial cells do not express additional anti-apoptotic proteins, such as Mcl-1 and Bcl-2, like other cell types. No other cell types were found to require Bcl-xL for survival during KSHV latent infection, likely due to the fact that they express several Bcl-2 family anti-apoptotic proteins. It is unlikely that the virus has evolved to induce apoptosis itself, rather it is more likely that the virus is inducing some host cell change that results in the activation of apoptosis. I found that the KSHV latent locus alone is sufficient to induce cell death in the absence of Bcl-xL. This led me to investigate if there was a specific KSHV latent gene that was inducing apoptosis during KSHV infection. I found that the kaposins, but no other KSHV latent genes or microRNAs (miRNAs), are necessary to render Bcl-xL necessary for survival. In addition to Bcl-xL, I sought to validate other top hits from the previous CRISPR/Cas9 screen. I examined the requirement of several genes for the survival and proliferation of KSHV latently infected cells, and determined that CYP27A1, a sterol hydroxylase, is needed for the proliferation of KSHV infected endothelial cells. Following the introduction and background (chapter 1) and materials and methods (chapter 2), chapter 3 details the validation of the top hit from our CRISPR/Cas9 screen, Bcl-xL. Chapter 4 describes how KSHV induces apoptosis during latent infection. In chapter 5, I validate the necessity of three other genes, Cyp27a1, Ylpm1, and Cmklr1, and determine that CYP27A1 is required for the proliferation of KSHV latently infected cells. Together this work identifies promising potential therapeutics for the specific treatment of Kaposi’s Sarcoma, improves our understanding of how KSHV changes the host cell during latent infection, and provides insight into the accuracy of global CRISPR/Cas9 screens.