Characterization of the impact of KSHV latent infection on endothelial cell lipid metabolism - Abstract: Characterization of the impact of KSHV latent infection on endothelial cell lipid metabolism. Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS), a malignancy with significant morbidity and mortality in southern, eastern, and central Africa, and in people living with HIV worldwide1,2. As a herpesvirus, KSHV has both lytic and latent replication cycles, and KS is thought to originate from latent KSHV infection of endothelial cells3–6. As there is no current therapeutic method targeting latent infection of any herpesvirus, a permanent cure for KS is nonexistent and current treatment relies on standard chemotherapy regimens. Our lab seeks novel therapeutic avenues by identifying pathologic changes in endothelial cells following latent KSHV infection that are pharmacologically targetable and upon which the survival of latently infected cells relies. One such alteration identified in our lab is KSHV’s rewiring of endothelial cell lipid metabolism. A previously published metabolomics screen from our lab reported that two- thirds of the long chain fatty acids detected in the screen were increased in abundance at 48 and/or 96 hours post-infection, while none were decreased in abundance, suggesting that fatty acid synthesis (FAS), the metabolic pathway by which endogenous saturated and monounsaturated fatty acids are produced de novo, is upregulated during latent infection7,8. This investigation further revealed that cytoplasmic lipid droplet (LD) formation is increased during latent infection, and that FAS is essential for endothelial cell survival during latency, which we have since further confirmed using denifanstat, an inhibitor of FAS currently in phase III clinical trials7,9. Though this increase in LD formation was presumed to be a by-product of the increase in FAS, we have since shown that LD formation is also essential to support cell survival. We now know that expression of the KSHV major latent locus is sufficient to induce LD formation, and that the latent viral protein vFLIP and the viral microRNAs are essential for this induction to occur, but the viral and cellular mechanism of this induction has yet to be characterized. Also unknown is why KSHV latently infected endothelial cells require FAS for survival, or how KSHV latent infection impacts exogenous lipid uptake. In this proposal, I am further investigating the viral and cellular mechanism of lipid droplet formation by overexpressing latent viral factors, identifying the source of the lipids constituting the lipid droplets, and evaluating whether vFLIP’s functions in NF-κB and AKT signaling are involved in this phenomenon10,11. I am also conducting lipidomics via LC-MS/MS to map the changes to the endothelial cell lipidome during latent KSHV infection, detect changes in lipid uptake, identify lipid species that may underpin the requirement of FAS for cell survival, and evaluate the impacts of lipid uptake and LD formation on sensitization to ferroptosis during latency. Together, completion of the work proposed will fill key gaps in knowledge regarding the specific impact of the latent KSHV replication cycle on lipid metabolism in endothelial cells, informing future therapeutic strategies for this disease.
