Functional analysis of the KSHV ORF57 protein in mRNA nuclear export mechanisms

Abstract

Kaposi's sarcoma-associated Herpesvirus (KSHV; HHV -8) is associated with multiple malignancies, including Kaposi's sarcoma. KSHV has two distinct life cycle phases, latent persistence and lytic replication. In contrast to other oncogenic Herpesviruses, lytic replication plays an important part in tumourigenicity and pathogenesis of KSHV. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. This in turn may lead to novel therapies, which could become an important strategy for the treatment of KSHV -associated diseases. Post-transcriptional regulation of RNA biogenesis is fundamental to KSHV lytic gene expression. The KSHV ORF57 protein plays an essential role in viral RNA processing, transcription, splicing, mRNA stability, nuclear mRNA export and translation. To date, it is unknown how ORF57 co-ordinates these many roles. Functional diversity of a protein can be achieved by post-translational modifications. We demonstrate that ORF57 is post-translationally methylated and inhibition of methylation has a dramatic effect on the ability of ORF57 to export intronless viral RN A out of the nucleus. This work shows that hypomethylation of ORF57 enhances its ability to bind RNA. Attempts were made to find ORF57 residues which are methylated and results identified PRMT5, a cellular protein methyltransferase as well as the putative demethylase MINA, which interact with ORF57. Furthermore, a proteomic-based approach was used to identify ce~lular proteins which are changed in their intracellular distribution or abundance upon ORF57 expression. This SILAC-based approach highlighted proteins and pathways affected by ORF57. Data showed changes in RNA processing pathways previously unknown to be affected by ORF57, e.g. mRNA polyadenylation and nucleoskeleton rearrangements. In addition, changes of putative, novel components of the human TREX complex were found. Data presented will provide a valuable resource not only to enhance our understanding of this viral pathogen but also to show new routes for drug intervention.EThOS - Electronic Theses Online ServiceGBUnited Kingdo