vGPCR-mediated ANO1 Modulation of Apoptosis Regulates Lytic KSHV Infection

ABSTRACT 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.