Arginine methylation regulates the function of a key herpesvirus nucleocytoplasmic shuttle protein

mRNA is transcribed in the nucleus and must be exported from the nucleus into the cytoplasm to get translated into a protein. In the nucleus, the RNA has to undergo a series of processing events prior to export and these are performed by a number of multi-protein complexes. However, how these complexes are recruited to the RNA and how each multi-protein complex is regulated to perform its specific function at the correct time is unknown. Herein, we aim to investigate how the function of one of these RNA binding multi-protein complexes is regulated using a viral model. We have previously demonstrated that the herpesvirus ORF57 protein is important for exporting herpesvirus mRNA out of the nucleus to get translated. ORF57 functions by binding to the herpesvirus RNA and specifically recruiting cellular proteins onto the viral RNA, which are essential for its correct processing and export. This system has uniquely identified that a multi-protein complex called hTREX is the only cellular protein complex which is required by ORF57 for herpesvirus mRNA nuclear export. Moreover, we have demonstrated that once hTREX is bound by ORF57 to the herpesvirus mRNA, the whole complex is transported through a domain within the nucleus called the nucleolus, before it is exported from the nucleus into the cytoplasm. Therefore, ORF57 is a multifunctional protein which sequentially, binds the viral RNA, then recruits hTREX to the viral RNA, then transports this complex to the nucleolus, then allows the complex to exit the nucleus. But what tells ORF57 to perform its many functions in the correct manner is unknown. This functional diversity of a protein can be controlled by different chemical modifications to the protein, such as adding or taking away a phosphate, acetate or methyl group on the protein. Interestingly, we have demonstrated that the herpesvirus ORF57 protein undergoes one such modification. We have shown that ORF57 can be methylated at arginine residues. Therefore, we aim to determine if adding or taking away methyl groups affects ORF57's function and in turn regulates the multi-protein complex responsible for exporting viral mRNA from the nucleus. To assess the role of methylation we will first identify which residues of ORF57 are methylated. We will then mutate these, so that ORF57 cannnot undergo methylation. This will then allow us to assess what effect it has upon the functioning of the ORF57 protein. Secondly, we will identify which cellular enzymes are responsible for performing this post-translational modification on the viral ORF57 protein. Finally, we have preliminary data to suggest that taking away the methyl group on ORF57 (or demethylating ORF57), is the trigger to tell ORF57 to exit the nucleus. We believe this happens in the nucleolus and we will identify which enzyme in the nucleolus performs this function. These data will provide a better understanding of how multi-protein complexes function to enable RNAs to exit the nucleus and also provide a better understanding of how viruses function in order to identify new antiviral targets in the longer term.

Processing of mRNA from transcription to translation is regulated by several multi-protein complexes. These multi-protein complexes are coupled to one another allowing processing events to occur in a strict temporal manner. However, how these complexes are regulated is unknown. We have utilised a viral model to study the function of one of these multi-protein complexes, which is involved in mRNA nuclear export. KSHV ORF57 orchestrates the assembly of an export-competent viral ribonucleoprotein particle (RNP) by recruitment of the hTREX complex to intronless viral mRNAs. Moreover, we have shown that the ORF57-mediated RNP trafficks through the nucleolus and this is essential for efficient nuclear export. How ORF57 co-ordinates it's many is unknown, but we have preliminary data to suggest that the methylation status of ORF57 is important for its function. Specifically, we have demonstrated that ORF57 can be methylated but also interacts with a nucleolar demethylase enzyme, suggesting that it can also undergo demethylation. Moreover, nucleocytoplasmic shuttling of ORF57 is regulated by its methylation status, as inhibition of ORF57 methylation redistributes ORF57 into the cytoplasm. We now aim to further investigate these observations and determine the role of arginine methylation in the functioning of this key herpesvirus nucleocytoplasmic shuttle protein. We will initially aim to identify the residues within ORF57 which are methylated using mass spectroscopy. Moreover, we will identify the enzymes which are responsible for both the methylation and demethylation of the ORF57 protein. An ORF57 mutant protein will then be produced which cannot undergo methylation. This mutant will then be compared to the wild type ORF57 protein to assess what effect arginine methylation / demethylation has upon the multifunctional roles of ORF57 such as RNA binding, hTREX recruitment, nucleolar trafficking, nucleocytoplasmic shuttling and intronless mRNA export.