Kaposi’s Sarcoma Herpes Virus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS) which is known to cause metabolic stress in infected host cells. KSHV reprograms host metabolic pathways for efficient viral replication and infectious virion production. Here, we report a time-course global metabolomics study conducted in the doxycycline-inducible iSLK.BAC16 cells to compare latent and lytic KSHV infection. Our data show that amino acid, central carbon, and nucleotide metabolic pathways are highly dysregulated upon reactivation to lytic replication. During lytic KSHV infection, pathway enrichment analysis shows that the top two most significantly impacted and dysregulated pathways are purine and pyrimidine metabolism. Further experiments have shown that nucleotide metabolism is required during lytic KSHV infection to produce maximal infectious virus. Treatment with the FDA-approved drug, methotrexate (MTX), a folate antagonist that inhibits cellular DHFR and decreases nucleotide metabolism by reducing tetrahydrofolate cofactors, significantly reduced KSHV late lytic viral gene expression upon reactivation compared to control. Additionally, titering cell-free supernatants from MTX-treated lytic KSHV-infected cells showed a significant reduction in infectious virion production. Furthermore, by adding folinic acid (FA), a downstream metabolite of the MTX-DHFR inhibition step, in the presence of MTX, late lytic gene expression and infectious virion production were significantly rescued. Furthermore, we observed a significant decrease in viral titer of murine herpesvirus 68 (MHV-68), a model virus to study gammaherpesvirus, after MTX treatment. Overall, our study demonstrates that metabolic inhibition during lytic gammaherpesvirus infection decreases productive infection and hence, serves as a potential therapeutic antiviral target.

Kaposi’s Sarcoma Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), which induces metabolic stress in infected host cells. KSHV reprograms host metabolic pathways for efficient viral replication and infectious virion production. Here, we report a time-course global metabolomics study conducted in iSLK.BAC16 cells to compare latent and lytic KSHV infection. Our data show that amino acid, central carbon, and nucleotide metabolic pathways are highly dysregulated upon reactivation. During lytic KSHV infection, pathway enrichment analysis identifies purine and pyrimidine metabolism as the top two most significantly impacted and dysregulated pathways. Further experiments have shown that nucleotide metabolism is required during lytic KSHV infection to produce maximal infectious virus. Treatment with the FDA-approved drug, methotrexate (MTX), a folate antagonist that decreases nucleotide metabolism by reducing tetrahydrofolate cofactors, significantly reduced KSHV copy number and late lytic viral gene expression upon reactivation compared to controls. Additionally, titers of cell-free supernatants from MTX-treated lytic samples showed a significant reduction in infectious virion production. Furthermore, MTX significantly decreased the viral titer of murine herpesvirus 68 (MHV-68), a model virus to study gammaherpesvirus. Overall, our study demonstrates that metabolic inhibition during lytic gammaherpesvirus infection decreases productive infection and hence serves as a potential therapeutic antiviral target.

Kaposi’s Sarcoma herpesvirus (KSHV) is an oncogenic, enveloped double-stranded DNA virus that causes Kaposi’s Sarcoma (KS), an endothelial-derived tumor that primarily affects immunocompromised individuals. KSHV persists in a biphasic lifecycle of latency and lytic reactivation, with the lytic phase driving viral dissemination and disease progression through expression of viral proteins that reprogram host survival pathways. One such protein is the viral G protein-coupled receptor (vGPCR), a constitutively active lytic gene product that promotes angiogenic and pro-survival signaling, although the host mediators of these effects remain incompletely defined. Here, we identify Anoctamin-1 (ANO1), a calcium-activated chloride channel with ant-apoptotic functions, as a downstream target of vGPCR in endothelial cells. RNA sequencing and RT-qPCR in vGPCR-expressing HMEC-1 cells revealed broad transcriptional reprogramming, including strong ANO1 induction. Functional studies showed that ANO1 depletion had little effect on viability in full-serum conditions but selectively sensitized vGPCR-expressing cells to apoptosis during serum starvation. This loss of viability was rescued by the pan-caspase inhibitor Z-VADFMK, indicating caspase-dependent apoptosis. To examine relevance during infection, we analyzed iSLK.BAC16 cells undergoing lytic reactivation. ANO1 was similarly upregulated during reactivation. Genetic knockdown or pharmacological inhibition of ANO1 with DES or Ani9 increased late-stage apoptosis while minimally affecting early apoptosis. Notably, ANO1 loss enhanced early and late lytic gene expression and increased production of infectious virions. These findings identify ANO1 as a vGPCR-induced host survival factor that suppresses apoptosis while modulating KSHV lytic replication, revealing a host ion-channel-dependent checkpoint linking cell survival to viral replication and KSHV pathogenesis. IMPORTANCE: KSHV drives tumorigenesis by manipulating host cell survival pathways, yet how apoptosis is regulated during infection remains incompletely understood. We identify ANO1 as a host factor induced by the vGPCR that suppresses caspase-dependent apoptosis in endothelial cells. ANO1 is upregulated in both vGPCR-expressing endothelial cells and during KSHV lytic reactivation in iSLK.BAC16 cells. Disruption of ANO1 increases apoptotic cell death and is accompanied by elevated lytic gene expression and greater production of infectious virions, suggesting that apoptosis can coincide with enhanced lytic replication. These findings implicate host ion-channel signaling in the control of apoptosis during KSHV infection and indicate that the balance between cell survival and apoptosis influences viral gene expression and replication. Understanding how KSHV manipulates host survival pathways provides insight into mechanisms governing viral replication and may inform approaches to limit KSHV-associated disease.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi’s sarcoma (KS), an endothelial cell-derived malignancy that primarily affects immunocompromised individuals. During lytic reactivation, KSHV expresses viral proteins that promote viral replication and modulate host signaling pathways. Here, we identified the calcium-activated chloride channel ANO1 (TMEM16A) as a previously unrecognized host factor induced by the KSHV lytic protein vGPCR. RNA sequencing, RT-qPCR, and immunofluorescence analyses demonstrated robust ANO1 upregulation in vGPCR-expressing endothelial cells and during KSHV lytic reactivation. siRNA-mediated depletion of vGPCR significantly reduced ANO1 expression, demonstrating that vGPCR contributes to ANO1 induction during infection. Functionally, ANO1 knockdown sensitized vGPCR-expressing endothelial cells to caspase-dependent apoptosis under serum-starved conditions, which was rescued by the pan-caspase inhibitor Z-VAD-FMK. In reactivated iSLK.BAC16 cells, genetic or pharmacological inhibition of ANO1 increased late apoptosis, enhanced KSHV lytic gene expression, and promoted infectious virion production. Together, these findings identify ANO1 as a vGPCR-regulated host factor that promotes cell survival and modulates KSHV lytic replication, highlighting ANO1 signaling as a potential therapeutic target in KSHV-associated disease.