Michael Lagunoff

Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells

Kaposi’s sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. 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.

Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells Activates Hypoxia-Induced Factors

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiological agent of Kaposis sarcoma, a highly vascularized, endothelial-derived tumor. A direct role for KSHV-mediated induction of angiogenesis has been proposed based upon the nature of the neoplasia and various KSHV gene overexpression and infection model systems. We have found that KSHV infection of endothelial cells induces mRNA of hypoxia-induced factor 1α (HIF1α) and HIF2α, two homologous alpha subunits of the heterodimeric transcription factor HIF. HIF is a master regulator of both developmental and pathological angiogenesis, composed of an oxygen-sensitive alpha subunit and a constitutively expressed beta subunit. HIF is classically activated posttranscriptionally with hypoxia, leading to increased protein stability of HIF1α and/or HIF2α. However, we demonstrate that both alpha subunits are up-regulated at the transcript level by KSHV infection. The transcriptional activation of HIF leads to a functional increase in HIF activity under normoxic conditions, as demonstrated by both luciferase reporter assay and the increased expression of vascular endothelial growth factor receptor 1 (VEGFR1), an HIF-responsive gene. KSHV infection synergizes with hypoxia mimics and induces higher expression levels of HIF1α and HIF2α protein, and HIF1α is increased in a significant proportion of the latently infected endothelial cells. Src family kinases are required for the activation of HIF and the downstream gene VEGFR1 by KSHV. We also show that KS lesions, in vivo, express elevated levels of HIF1α and HIF2α proteins. Thus, KSHV stimulates the HIF pathway via transcriptional up-regulation of both HIF alphas, and this activation may play a role in KS formation, localization, and progression.

Viral Activation of Cellular Metabolism

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for the replication of each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways.

Global Metabolic Profiling of Infection by an Oncogenic Virus: KSHV Induces and Requires Lipogenesis for Survival of Latent Infection

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposis Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposis 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.

Persistent Activation of STAT3 by Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposis sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castlemans disease. STAT3 has been shown to be important for the maintenance of primary effusion lymphoma cells in culture and is chronically activated in many tumor cell lines. However, little is known about the role of KSHV in the activation of STAT3 or the role of STAT3 in KS tumors. We demonstrate that STAT3 is activated by KSHV infection of endothelial cells, the KS tumor cell type, in a biphasic fashion. Viral binding and entry activate STAT3 in the first 2 h after infection, but this activation dissipates by 4 h postinfection. By 12 h after KSHV infection, concomitant with the expression of latent genes, STAT3 is once again activated, and this activation persists for as long as latent infection is maintained. Activated STAT3 translocates to the nucleus, where it can bind to STAT3-specific DNA elements and can activate STAT3-dependent promoter activity. Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. KSHV upregulates gp130 receptor expression, and both gp130 and JAK2 are required for the activation of STAT3. However, neither human nor viral interleukin-6 is required for STAT3 activation. Persistent activation of the oncogenic signal transducer, STAT3, by KSHV may play a critical role in the viral pathogenesis of Kaposis sarcoma, as well as in primary effusion lymphomas.

Dengue Virus Induces and Requires Glycolysis for Optimal Replication

ABSTRACT Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. IMPORTANCE Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens.

Activation of Akt through gp130 Receptor Signaling Is Required for Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphatic Reprogramming of Endothelial Cells

ABSTRACT Kaposis sarcoma (KS) is the most common tumor of AIDS patients worldwide. KS-associated herpesvirus (KSHV) is the infectious cause of this highly vascularized skin tumor. The main cell type found within a KS lesion, the spindle cell, is latently infected with KSHV and has markers of both blood and lymphatic endothelial cells. During development, lymphatic endothelial cells differentiate from preexisting blood endothelial cells. Interestingly, KSHV infection of blood endothelial cells induces lymphatic endothelial cell differentiation. Here, we show that KSHV gene expression is necessary to maintain the expression of the lymphatic markers vascular endothelial growth factor receptor 3 (VEGFR-3) and podoplanin. KSHV infection activates many cell signaling pathways in endothelial cells and persistently activates STAT3 through the gp130 receptor, the common receptor of the interleukin 6 family of cytokines. We find that KSHV infection also activates the phosphatidylinositol 3-OH-kinase (PI3K)/Akt cell signaling pathway in latently infected endothelial cells and that gp130 receptor signaling is necessary for Akt activation. Using both pharmacological inhibitors and small interfering RNA knockdown, we show that the gp130 receptor-mediated activation of both the JAK2/STAT3 and PI3K/Akt cell signaling pathways is necessary for KSHV-induced lymphatic reprogramming of endothelial cells. The induction of the lymphatic endothelial cell-specific transcription factor Prox1 is also involved in KSHV-induced lymphatic reprogramming. The activation of gp130 receptor signaling is a novel mechanism for the differentiation of blood endothelial cells into lymphatic endothelial cells and may be relevant to the developmental or pathological differentiation of lymphatic endothelial cells as well as to KSHV pathogenesis.

Establishment and Maintenance of Kaposi's Sarcoma-Associated Herpesvirus Latency in B Cells

ABSTRACT Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is the infectious cause of Kaposis sarcoma and is also associated with two B-cell lymphoproliferative diseases, primary effusion lymphoma and the plasmablastic form of multicentric Castlemans disease. KSHV is also found in the B-cell fraction of peripheral blood mononucleocytes of some KS patients. Despite in vivo infection of B cells and the ability of KSHV to infect many cell types in culture, to date B cells in culture have been resistant to KSHV infection. However, as shown here, the lack of infection is not due to the inability of B cells to support latent KSHV infection. When KSHV DNA is introduced into B cells, the virus is maintained as an episome and can establish and maintain latency over the course of months. As in all primary effusion lymphoma cell lines, there is a low level of spontaneous lytic replication in latently infected BJAB cells. Importantly, viral gene expression is similar to that of primary effusion lymphoma cell lines. Furthermore, the virus can be reactivated to higher levels with specific stimuli and transmitted to other cells, indicating that this is a productive infection. Thus B cells in culture are capable of establishing, maintaining, and reactivating from latency. These studies provide a controlled system to analyze how KSHV alters B cells during KSHV latency and reactivation.

Vaccinia Virus Requires Glutamine but Not Glucose for Efficient Replication

ABSTRACT Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCE Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV requires exogenous glutamine for efficient replication, and inhibition of glutamine metabolism effectively blocks VACV protein synthesis. This study defines a major metabolic perturbation essential for the replication of a poxvirus and may lead to the discovery of novel antiviral therapies based on metabolic inhibitors.

The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro

ABSTRACT Kaposis sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposis sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.