Impact of a viral endoribonuclease on the nucleocytoplasmic compartmentalisation of the cellular transcriptome

Viruses infect human cells and exploit a wide range of cellular pathways in their replication strategies, using them to their own advantage to turn the cell in to a virus production. Many proteins produced by viruses are similar to cellular proteins and perform equivalent activities to help the virus to replicate. As such, these virus proteins can act as excellent models for as yet uncharacterised cellular activities and help to unravel complex pathways in the cell.

In the cell, RNA messages are made from genes in the nucleus and then transported to the cytoplasm to be translated in to proteins. Important enzymes called endoribonucleases are involved in regulating the levels of RNA message in the cell, to control the amount and specificity of protein being made at any one time. These enzymes have roles in diverse processes such as responses to stress or virus infection, or regulation of inflammation. As such, they are critical for maintaining a balanced cellular environment and keeping cells healthy. Moreover, mutations in a number of endoribonucleases have been linked to a range of diseases including inherited blood disorders, cancer and changes through ageing.

Many viruses including herpesviruses and influenza viruses also make endoribonucleases to regulate the proteins that are being made within the cell. This proposal concerns the herpes simplex virus endoribonuclease called vhs. We have found that this single virus protein has the ability to not only eliminate RNA messages, but also to entrap many of them in the nucleus such that they are produced but not available for translation in to protein, thereby efficiently regulating protein production in the cell.

At the molecular level, the vhs mode of action is similar to its range of cellular counterparts. Hence, using vhs as a model system, we aim to unravel the complex set of events initiated by vhs. In particular, we aim to use state-of-the-art technology to define how the nuclear population of RNA message changes on a global level in response to vhs activity. We will also investigate the mechanism of these vhs-induced changes by exploring its effect on export pathways from the nucleus to the cytoplasm. Finally, we will determine how the vhs protein itself is controlled within the cell to ensure that its expression is non-lethal to the cell.

This study will advance current understanding of the role of these enzymes in the regulation of protein production in both virus infected and healthy cells. Moreover, it will benefit other studies on diseases where these enzymes have been found to be defective, thereby impacting both fundamental and translational science.

The turnover of mRNA by ribonuclease degradation is critical for the regulation of gene expression in response to environmental changes in the eukaryotic cell, and must be carefully controlled. Many viruses express endoribonucleases that alter the transcriptomic landscape of the cell to favour translation of virus proteins, making them excellent models to reveal new understanding of endoribonucleases in general. The herpes simplex virus endoribonuclease called vhs regulates gene expression during infection by inducing the degradation of multiple cellular transcripts in the cytoplasm. We have recently shown that vhs also alters the relative nucleocytoplasmic compartmentalisation of the cellular transcriptome by inhibiting nuclear export. Using this virus enzyme as a paradigm for endoribonuclease activity, we aim to use RNAseq and mass spectrometry analyses to delineate the consequence of vhs expression on the global nuclear landscape and identify specific subsets of transcripts and proteins that undergo nuclear retention when vhs is expressed. We will also use cell biological techniques to determine the specific nuclear export receptors that its action may affect. In this way, we will determine the specificity of vhs action on the nucleus. Cells expressing GFP-tagged vhs will be utilised to establish the trafficking properties of vhs and to re-evaluate the key cellular proteins that interact with vhs. Finally, given that high levels of expression of this endoribonuclease are detrimental to the cell, we will also investigate the posttranscriptional regulation involved in maintaining this endoribonuclease at non-lethal levels by isolating the RNA binding proteins that interact with specific inhibitory sequences we have identified in the vhs transcript. These studies will impact current understanding of the role of endoribonucleases in the global regulation of mRNA levels, and underpin the activity of vhs and other endoribonucleases for virus infection.

The immediate goal of this research is to delineate the fundamental biology of the vhs endoribonuclease protein of HSV and as such, its short-term impact will be to advance scientific knowledge on how these proteins function in the cell and how they contribute to regulation of gene expression. The study is an interdisciplinary collaboration and will advance the fields of both herpesvirology and RNA biology, providing opportunities for impact through new collaborations, open access publications, and scientific presentations to conferences and invitation to other Institutions.

The project has the potential to impact on future research leadership, as the appointed PDRA will be trained in specialist transferable skills such as RNAseq, confocal microscopy and proteomics. This individual will help in the training of students in the laboratory, thereby gaining supervisory experience, and will be trained in various skills, including scientific writing and presenting skills, which will enhance their potential to become a research leader in the future. The PDRA will also develop networking skills through attendance at conferences.

These studies will also impact undergraduate and MSc students at the University of Surrey, with the incorporation of new data in to the lectures delivered by the PI and Co-I, and the opportunity to carry out research projects related to the project. In addition, our results will be communicated to the general public in an understandable and non-technical format through press release from the University of Surrey Media Centre.

In the longer term, this research has the potential to impact on the understanding of human disease that involves mutation in endoribonucleases such as SLFN14 in inherited thrombocytopenia or APE1 in cancer. It will also impact the development of new antiviral treatments for HSV infection. HSV is the causative agent of oral and genital herpes and the quality of life of individuals who suffer from frequent reactivation of HSV could be greatly improved by new anti-HSV treatment. The ability to block reactivation of HSV could greatly reduce the occurrence of both the frequent symptoms and the rarer but more serious outcomes of HSV infection such as encephalitis and keratitis.

This project could therefore ultimately contribute to the economic competitiveness of the UK through the development of drugs that target HSV or VZV, or treatments for animal alphaherpesvirus infections. Our discoveries would be commercialized by existing companies in the UK, with the potential for enhanced wealth generation and employment opportunities.