Identification of Targetable Vulnerabilities During Latent KSHV Infection

Abstract

Viruses are defined as obligate intracellular parasites that require host processes to replicate. Latent virus life cycles are no exception to this definition, as viruses are still reliant on host machinery for continued proliferation and maintenance of viral genomes, even in the absence of lytic replication. In this thesis, I used essentiality screening to identify host factors on which Kaposi’s Sarcoma Associated Herpesvirus (KSHV) relies for the proliferation and survival of latently infected cells. KSHV is the etiological agent of Kaposi’s Sarcoma (KS), an endothelial cell-based tumor where more than 90\% of the endothelial cells in the tumor are latently infected with KSHV. While traditional therapies for herpesviruses target lytic replication, the prevalence of latency in KS necessitates exploration of options for intervening in this stage of the viral life cycle. I performed CRISPR/Cas9 screening using lentiviral vectors encoding a library of single guide RNAs (sgRNAs) targeting every protein coding gene in the human genome. I compared mock infected and KSHV infected endothelial cells eight days post infection to identify genes essential to latent KSHV infection. Additional sub-pool screening was carried out targeting genes from the initial whole genome screen to validate the results. Among the list of hits, were a number of genes involved in mitochondrial translation. This is a reasonable pathway for chemical inhibition, as mitochondrial ribosomes are sensitive to antibiotics targeting bacterial ribosomes due to their shared ancestry. I found that treatment of latently infected endothelial cells results in suppression of cellular proliferation relative to mock infected controls. Additionally, antibiotic treatment of KSHV latently infected primary effusion lymphomas (PELs) induces cell death. I also found that inhibition of respiration, either chemically or by eliminating mitochondrial genomes from cells, results in suppressed proliferation and cell death. The dependence on mitochondrial function led me to characterize the status of mitochondria during KSHV infection. I found that latent KSHV infection leads to more interconnected mitochondrial networks, higher mitochondrial transcript levels, and increased mitochondrial genome copies. In attempting to determine how KSHV facilitates mitochondrial changes, I found that three latent proteins localize to the mitochondria. In experiments following up on a metabolomic screen carried out by our lab, I found that the latent locus of KSHV is essential for accumulation of lipid droplets during infection. I also found that lipid droplet formation is essential for cell survival during latent infection, but that this requirement is lost during infection with a mutant virus lacking the latent miRNA cluster. This work improves our understanding of the role of mitochondrial function in the persistence of latently infected cells and provides possible specific therapeutic targets for the main proliferating cells in KS tumors.