The effect of Kaposi’s sarcoma-associated herpesvirus RTA expression upon the cellular proteome

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS). Like all herpesviruses, KSHV has a bi-phasic life cycle, with a dormant latent phase and a productive lytic phase. The switch from viral latency to lytic replication is mediated by the replication and transcription activator protein (RTA). RTA activates KSHV lytic gene expression via direct and indirect binding to lytic promoters. Moreover, it functions as an E3 ubiquitin ligase, actively degrading repressor proteins, such as Hey1, maintaining the virus in the latent state.

The first aim of this study was to determine if RTA functions as a SUMOylation targeted ubiquitin ligase (STUbL), which recognises poly-SUMOylated targets via SUMO interacting motifs (SIMs). Results presented herein demonstrate that the Hey1 repressor protein is SUMO2 modified. Furthermore, mutation of SIM domains within RTA resulted in attenuation of RTA-mediated degradation of Hey1 and lytic reactivation. However, SIM mutation also severely reduced RTA-mediated transactivation. These results suggest that RTA may have STUbL activity but additional work is required to reconcile the effect of SIM mutation upon transcriptional activity.

The second aim of this investigation was to identify novel points of interaction between RTA and the host cell. In chapter 4, SILAC-based quantitative proteomics identified hundreds of proteins which demonstrated a significant change in abundance upon RTA expression. Abundance of the cellular protein ARID3B was found to increase over 7-fold in the nuclear fraction. Furthermore, ARID3B was shown to re-localise to viral replication centres upon lytic reactivation. In chapter 5 SILAC-based immunoprecipitations were performed to identify novel RTA interaction partners. The cellular co-activator RBM14 was found to be enriched in two independent SILAC data sets and subsequent investigation demonstrated that RBM14 localisation was altered upon RTA expression. These novel observations highlight the potential significance of cellular factors in KSHV infection. Further investigation is required to fully characterise the role of these proteins in viral reactivation.