Egr-1 regulates RTA transcription through a cooperative involvement of transcriptional regulators

Abstract

Kaposi's sarcoma associated herpesvirus (KSHV) regulates the host cellular environment to establish life-long persistent infection by manipulating cellular signaling pathways, with approximately 1- 5% of cells undergoing lytic reactivation during the course of infection. Egr-1 (Early Growth Response Factor-1) is one such cellular transcription factor, which gets phosphorylated during the lytic phase of viral life cycle to perpetrate its function. This study demonstrates the mechanism of how Egr-1 mediates transcription of the immediate early gene, RTA (Replication and transcription activator), which is the lytic switch gene of KSHV. Egr-1 depleted KSHV infected cells exhibited reduced expression of RTA. Also, an increase in Egr- 1 phosphorylation led to a higher virion production, which was suppressed in the presence of p38 and Raf inhibitors. Reporter assays showed that coexpression of Egr- 1 and CBP (CREB-binding protein) enhances RTA promoter activity as compared to the expression of either Egr-1 or CBP alone. Binding of Egr-1 and CBP at RTA promoter was analyzed by chromatin immunoprecipitation assay (ChIP), which showed an enhanced accumulation during viral reactivation. Mutation in Egr-1 binding site of the RTA promoter eliminated Egr-1 response on promoter activation. Furthermore, de novo infection of THP-1 (monocytic) and HUVECs (endothelial) cells showed an upregulation of Egr-1 phosphorylation, whereas depletion of Egr-1 reduced the mRNA levels of RTA during primary infection. Together, these results demonstrate a cooperative role of Egr-1 and CBP in mediating RTA transcription, which significantly improves our understanding of the involvement of cellular factors controlling RTA transcription in KSHV pathogenesis.