Virus manipulation of host non-coding RNA regulatory networks

Lead Research Organisation: University of Leeds
Department Name: Sch of Molecular & Cellular Biology

Abstract

It has been known for some years that only ~2% of the human genome encodes for proteins, whereas ~80% is actively transcribed into RNAs with no obvious coding capacities, known as non-coding RNAs (ncRNAs). The importance of this non-coding transcriptome is emphasised due to the fact that ncRNAs are frequently altered in many human diseases, including aging, cancer, auto-immunity and infections. Functional studies have shown that ncRNAs are critical regulators of gene expression. For example, a group of short ncRNAs known as microRNAs (miRNAs), modulate gene expression by binding to their target protein coding transcript, which leads either to the transcript's translation repression, cleavage or decay. As such, research in this area has mainly focussed on identifying the interaction between specific miRNAs and their target transcript(s). However, emerging evidence now suggests the existence of an unexpected interplay between different ncRNAs that strongly influences, for example, how a miRNA can bind to its target. In this case, other ncRNA species, such as circular RNAs and other long ncRNAs, function as competing endogenous RNAs, interacting with miRNAs to sponge or decoy the miRNA, thus inhibiting the miRNA from binding to the target mRNA and preventing its repression. As such, this network of ncRNA-ncRNA interactions can have a profound effect on the regulation of gene expression in many cellular processes.

We have exciting preliminary data suggesting that a herpesviruses has evolved ways to manipulate these ncRNA regulatory networks to enhance virus gene expression and modulate the host response to infection. We have demonstrated that during herpesvirus infection several circular RNAs are upregulated whereas the majority of dysregulated miRNAs are downregulated. This suggests that these virus-induced circular RNAs could sponge specific miRNAs to outcompete their binding to target protein coding transcripts and prevent their repression or degradation.

We now aim to further investigate these observations and identify the interplay between ncRNA species and their associated regulatory networks which are manipulated by the virus. Furthermore, we will determine why these regulatory networks are altered during infection by determining the role of the target protein-coding transcripts that are aberrantly expressed due to the manipulation of their respective networks. Moreover, we will investigate novel mechanisms of how a virus enhances circular RNA levels during infection. Finally, we have identified a group of miRNAs that in contrast to the majority of downregulated miRNAs are actually increased during infection, which suggests the virus enhances their production to repress or degrade cellular transcripts which are probably detrimental to virus infection. We will determine how the viruses upregulates these miRNAs and also determine the inhibitory role of their target mRNAs in virus replication.

In summary, this project will identify novel ways a virus can manipulate the host cell to enhance its own replication and provide a better understanding how the interplay between different ncRNA species can regulate gene expression. A better knowledge of these fundamental processes has the potential for far reaching impacts on our understanding of cell and developmental biology processes, the development of human disease and provide new strategies for therapeutic interventions of important human pathogens.

Technical Summary

Non-coding RNA (ncRNAs) constitute the majority of the human transcriptome. ncRNAs play diverse roles in a multitude of cellular processes, functioning as critical regulators of gene expression. This is reinforced by ncRNA dysregulation being implicated in the development and progression of a wide range of human diseases. To date research into the mechanisms of ncRNA function has mainly focussed on the unidirectional regulation of target protein-coding transcripts, particularly by miRNAs. However, it is now evident that there exists an unexpected interplay between different ncRNA species, that strongly influence how gene expression is regulated. This hidden cross-talk of ncRNA-ncRNA interactions forms competitive regulatory networks. Notably, aberrant expression of any network component could derail the complex regulatory circuit, culminating in the development and progression of disease.

We have exciting preliminary data to suggest Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates ncRNA regulatory networks to enhance virus gene expression and modulate the host response to infection. As such, this provides an excellent model to study how dysregulation of these ncRNA-based networks impact on host gene expression.

The aim of the project will determine how and why these ncRNA regulatory networks are altered during infection. We will investigate novel mechanisms of how a virus dysregulates ncRNA biogenesis during infection and determine the role of the target protein-coding transcripts aberrantly expressed due to virus-mediated manipulation of these ncRNA networks.

In summary, this project will identify novel ways viruses manipulate the host cell to enhance their replication and provide a better understanding of how the interplay between different ncRNA species can regulate gene expression and how this impacts on human disease. Moreover, it may provide new strategies for therapeutic interventions of important human pathogens.

Planned Impact

The proposal builds upon previous novel work which has focussed on applying omic-based strategies to understanding the interactions between viruses and the host cell. The aim of this current proposal is to test the hypothesis that herpesvirus infection manipulates ncRNA regulatory networks. In particular, we aim to examine how a virus commandeers the intricate interplay between ncRNAs to enhance virus gene expression and modulate the host response to infection.

Whilst this study is fundamental in nature, the impact of the research will be wide reaching. The virus model provides an excellent tool to determine how ncRNA regulatory networks exert regulatory control on gene expression. Aberrant ncRNA processing and levels are implicated in a number of human diseases, therefore any clues as to how cellular ncRNA processing quality control checkpoints are bypassed by virus infection generated from this project will be of interest to the pharmaceutical industry. Recent advances of biological drugs have broadened the scope of therapeutic targets for a variety of human diseases. This holds true for RNA-based therapeutics, recent developments in this area have improved synthetic delivery carriers and chemical modifications to enhance their stability. These emerging drug approaches will be key in modulating ncRNA regulatory networks which are dysregulated in human diseases.

In addition, a key element of this project is the characterisation of essential virus-host cell interactions which will provide avenues for novel antiviral strategies. As numerous virus-host cell interactions are conserved in herpesviruses this approach may have generic applications for the treatment of a variety of additional human and animal diseases caused by this large family of viruses. Therefore, these discoveries may foster new collaborations with the pharmaceutical and other commercial industries to exploit these findings for new therapeutic strategies.

In the longer term, exploitation of these findings by the commercial sector may lead to new treatments for a wide range of diseases and virus infections, and this will provide benefits to the quality of life of the general public. Moreover, exploitation of the research findings by the commercial sector is also likely to have a direct impact on the prosperity of the general public of the UK, through increased investment and employment opportunities that will arise from new therapeutic drugs.
 
Description Non-coding RNAs (ncRNAs) constitute the majority of the human transcriptome. Tremendous interest in the field has delineated many aspects of ncRNA function, showing they are critical regulators of gene expression involved in many cellular pathways. However to date, research has mainly focused on the unidirectional ncRNA-mediated regulation of target protein-coding transcripts, particularly by miRNAs. Emerging evidence now suggests the existence of an unexpected interplay amongst ncRNAs that strongly influences how gene expression is regulated. This intricate network of ncRNA:ncRNA interactions has the potential to modulate gene expression involved in multiple cellular pathways. As such, dysregulation of ncRNA regulatory networks may impact on the development and progression of a wide range of diseases.

In this grant we have shown that the oncogenic herpesvirus, KSHV, dysregulates a host cell ncRNA regulatory network to enhance its own replication. Central to this dysregulation is the virus-mediated upregulation of a host cell circRNA, circHIPK3. We then demonstrate that circHIPK3, functions as a competing endogenous RNA to regulate the miR-30c/DLL4 axis. Importantly, dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.

In summary, our research describes a new and exciting advancement in the field, demonstrating dysregulation of a host cell ncRNA regulatory network is a direct contributor to KSHV lytic replication. These findings highlight the essential nature of this network and potential for therapeutic intervention for this important human pathogen.
Exploitation Route Novel therapeutic strategies for virus infections
Sectors Pharmaceuticals and Medical Biotechnology

 
Description We are determining how virus can manipulate non-cosing RNA pathways to enhance their own replication - we are identifying new cellular pathways which are essential for virus replication which could be developed into new therapeutic strategies
First Year Of Impact 2020
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description International collaboration 
Organisation Rhodes University
Country South Africa 
Sector Academic/University 
PI Contribution Dr Adrienne Edkins, Rhodes University, South Africa
Collaborator Contribution Investigating host cell - virus interactions focussing on molecular chaperones
Impact Kirigin. E., Ruck, D.K., Jackson, Z., Murphy, J., McDonnell, E., Okpara, M.O., Whitehouse, A. & Edkins, A.L. (2020). Regulation of Kaposi's sarcoma-associated herpesvirus biology by host molecular chaperones. Book Chapter.
Start Year 2017
 
Description International collaboration - Whitehouse Schneekloth 
Organisation National Cancer Institute
Country Lithuania 
Sector Hospitals 
PI Contribution We have identified specific viral mRNAs that are m6A modified resulting in structural changes which implies that inhibitory small molecules may be able to differentiate between the normal and modified forms.
Collaborator Contribution To identify inhibitory RNA-binding small molecules which selectively bind the ORF50 stem loop, in its normal and modified forms, Prof Schneekloth has performed a Small Molecule Microarray Screen (SMMS), encompassing 22,828 drug-like molecules. This identified the following small molecules: 14-bind 'N' only; 40-bind 'M' only and 13-bind both 'N' and 'M' forms. Excitingly, we show that addition of these compounds, at non-cytotoxic concentrations, can effectively reduce KSHV lytic replication and infectious virion production. These results suggest that an RNA-binding small molecule which selectively binds the m6A-modified ORF50 stem loop can inhibit KSHV lytic replication
Impact None to date Multi-disiplinary - virology and RNA biology, medicinal chemistry
Start Year 2020
 
Description Be curious Leeds Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Be Curious is the University's annual, family-friendly, research open day, an event which is usually held on campus, but in 2020 we went virtual. Over 100 of our brilliant researchers were involved in creating lots of fantastic content - from 2 minute demonstrations to try at home activities, research-related digital jigsaws and cartoon strips - to help you explore just some of the exciting things we get up to at the University of Leeds.
Year(s) Of Engagement Activity 2020,2021,2022
URL http://www.leeds.ac.uk/info/4000/around_campus/460/be_curious_festival-about_leeds_and_yorkshire
 
Description Presentation at Beaver camp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk and experiment to extract DNA from fruit as local scout and beavers group in Leeds
Year(s) Of Engagement Activity 2017,2018,2019,2020,2022
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I do a talk about DNA and viruses and run an practical (extraction of DNA from fruit) and imaging of cheek cells to KS1 and KS2 (years 3,4,5,6) pupils at Bramhope primary school. Once a year as part of science week
Year(s) Of Engagement Activity 2016,2017,2018,2019,2020,2022