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Kaposi’s sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.

Kaposi’s Sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor common in AIDS patients and many countries in Africa. KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell, a cell expressing endothelial cell markers. To identify host genes important for KSHV latent infection of endothelial cells we previously used a global CRISPR/Cas9 screen to identify genes necessary for the survival or proliferation of latently infected cells. In this study we rescreened top hits and found that the highest scoring gene necessary for infected cell survival is the anti-apoptotic Bcl-2 family member Bcl-xL. Knockout of Bcl-xL or treatment with a Bcl-xL inhibitor leads to high levels of cell death in latently infected endothelial cells but not their mock counterparts. Cell death occurs through apoptosis as shown by increased PARP cleavage and activation of caspase-3/7. Knockout of the pro-apoptotic protein, Bax, eliminates the requirement for Bcl-xL. Interestingly, neither Bcl-2 nor Mcl-1, related and often redundant anti-apoptotic proteins of the Bcl-2 protein family, are necessary for the survival of latently infected endothelial cells, likely due to their lack of expression in all the endothelial cell types we have examined. Bcl-xL is not required for the survival of latently infected primary effusion lymphoma (PEL) cells or other cell types tested. Expression of the KSHV major latent locus alone in the absence of KSHV infection led to sensitivity to the absence of Bcl-xL, indicating that viral gene expression from the latent locus induces intrinsic apoptosis leading to the requirement for Bcl-xL in endothelial cells. The critical requirement of Bcl-xL during KSHV latency makes it an intriguing therapeutic target for KS tumors.

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi's Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.

Kaposi's Sarcoma Herpesvirus (KSHV) is present in the main tumor cells of Kaposi's Sarcoma (KS), the spindle cells, which are of endothelial origin. KSHV is also associated with two B-cell lymphomas, Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease. In KS and PEL, KSHV is primarily latent in the infected cells, expressing only a few genes. Although KSHV infection is required for KS and PEL, it is unclear how latent gene expression contributes to their formation. Proliferation of cancer cells occurs despite multiple checkpoints intended to prevent dysregulated cell growth. The first of these checkpoints, caused by shortening of telomeres, results in replicative senescence, where cells are metabolically active, but no longer divide. We found that human dermal lymphatic endothelial cells (LECs) are more susceptible to KSHV infection than their blood-specific endothelial cell counterparts and maintain KSHV latency to higher levels during passage. Importantly, KSHV infection of human LECs but not human BECs promotes their continued proliferation beyond this first checkpoint of replicative senescence. The latently expressed viral cyclin homolog is essential for KSHV-induced bypass of senescence in LECs. These data suggest that LECs may be an important reservoir for KSHV infection and may play a role during KS tumor development and that the viral cyclin is a critical oncogene for this process.

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.

Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients worldwide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi’s sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identify specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu ), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch.

Viruses manipulate numerous host factors and cellular pathways to facilitate the replication of viral genomes and the production of infectious progeny. One way in which viruses interact with cells is through the utilization and exploitation of the host lipid metabolism. While it is likely that most—if not all—viruses require lipids or intermediates of lipid synthesis to replicate, many viruses also actively induce lipid metabolic pathways to sustain a favorable replication environment. From the formation of membranous replication compartments, to the generation of ATP or protein modifications, viruses exhibit differing requirements for host lipids. Thus, while the exploitation of lipid metabolism is a common replication strategy, diverse viruses employ a plethora of mechanisms to co-opt these critical cellular pathways. Here, we review recent literature regarding the exploitation of host lipids and lipid metabolism specifically by DNA viruses. Importantly, furthering the understanding of the viral requirements for host lipids may offer new targets for antiviral therapeutics and provide opportunities to repurpose the numerous FDA-approved compounds targeting lipid metabolic pathways as antiviral agents.

Kaposi's Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi's Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells.

Kaposi's Sarcoma (KS), the most common tumor of AIDS patients, is a highly vascularized tumor supporting large amounts of angiogenesis. The main cell type of KS tumors is the spindle cell, a cell of endothelial origin, the primary cell type involved in angiogenesis. Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS and is likely involved in both tumor formation and the induction of angiogenesis. Integrins, and specifically integrin αVβ3, have known roles in both tumor induction and angiogenesis. αVβ3 is also important for KSHV infection as it has been shown to be involved in KSHV entry into cells. We found that during latent infection of endothelial cells KSHV induces the expression of integrin β3 leading to increased surface levels of αVβ3. Signaling molecules downstream of integrins, including FAK and Src, are activated during viral latency. Integrin activation by KSHV is necessary for the KSHV-associated upregulation of a number of angiogenic phenotypes during latent infection including adhesion and motility. Additionally, KSHV-infected cells become more reliant on αVβ3 for capillary like formation in three dimensional culture. KSHV induction of integrin β3, leading to induction of angiogenic and cancer cell phenotypes during latency, is likely to be important for KS tumor formation and potentially provides a novel target for treating KS tumors.