Kaposi’s sarcoma-associated herpesvirus replication and transcription activator regulates extracellular matrix signal pathway

Abstract

Kaposi’s Sarcoma (KS) is a malignancy caused by infection with Kaposi’s Sarcoma-associated Herpesvirus [KSHV; also known as Human Herpesvirus 8 (HHV8)] in which tumor cells show a characteristic ‘spindle-like’ morphology. The transcription factor RTA (Replication and Transcription Activator) is the viral protein responsible for reactivating KSHV from its latent state. Production of RTA in latently infected cells causes a number of viral proteins to be produced and leads to a cascade of gene expression changes in both viral and host genes. Previous work in our lab showed that RTA was capable of reprogramming cells in vitro to display a spindle-like morphology. In this study we aimed to identify the host gene expression changes caused directly by RTA which could be responsible for that reprogramming. To that end, Madin-Darby Canine Kidney cells (MDCK cells) were chosen as a model for KSHV-naïve mammalian cells. Differences in host gene expression levels in a culture of MDCK cells transfected with a plasmid coding for expression of RTA compared to MDCK cells transfected with a similar plasmid lacking the RTA gene were measured by whole transcriptome sequencing (RNA-Seq). Cells containing the RTA-coding plasmid adopted a spindle-like morphology and showed at least a two-fold change in expression level in approximately 180 genes. Those 180 genes were then screened for known associations to signaling pathways in order to determine which might be involved with the morphological changes observed and/or biological significance. The expression levels of the 10 genes identified by that screening were then verified by quantitative real time PCR (qPCR). Of those 10 genes, eight were identified as potentially associated with the morphological changes, including three genes associated with extra cellular matrix (ECM) destruction (MMP9, CTSD, and CTSS) that were down-regulated; two genes associated with blocking ECM destruction (TIMP1 and TIMP2) that were pregulated; two ECM component genes (LAMC2 and COL1A2) that were upregulated; and one gene associated with blocking cell-cell and cell-ECM adhesion (MUC1) that was downregulated. The remaining two genes (MAP2K1 and podoplanin) were identified as potentially biologically significant, but not directly involved in regulating morphology. MAP2K1 is associated with epithelial dedifferentiation and was down-regulated; and the lymphatic endothelial specific marker podoplanin (PDPN) was up-regulated. Taken together, the differences in morphology and gene expression between RTA-producing cells and controls suggest a possible role for RTA in the formation of the spindle cells that characterize Kaposi’s sarcoma.