Analysis of Dengue virus NS5 protein - host cell interactions.

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

Dengue virus (DENV) is a mosquito borne flavivirus that causes the most important arthropod-borne viral disease of humans. Dengue disease ranges from mild fever to the potentially fatal dengue haemorrhagic fever/ shock syndromes (DHF/DSS). It is estimated that at least 50 million dengue infections and 250000 cases of DHF occur annually. DHF is characterised by an increase in vascular permeability, which is believed to be immune mediated. Our current understanding of dengue disease is limited and there are no vaccines or antiviral treatments in use to control the spread of DENV.
Our research is aimed at understanding how DENV replicates and causes disease. The DENV NS5 protein has been found to be essential for replication of the virus and is an important target for antiviral drug development. Recent evidence also suggests that the NS5 protein may play a role in promoting disease. We intend to investigate how the NS5 interacts with the host cell to alter host cell processes, defining the regions of the NS5 protein involved and determining whether these features are common to different strains of DENV.
By determining how individual DENV proteins modulate host cell processes, we will increase our understanding of DENV disease. This knowledge can be used to develop improved DENV vaccines and antiviral agents that are urgently needed. In addition, the research proposed in this application is potentially relevant to our general understanding of how the immune system functions to control microbial disease.

Technical Summary

Dengue virus (DENV) is a mosquito borne flavivirus that causes the most important arthropod-borne viral disease of humans. Dengue disease ranges from mild fever to the potentially fatal dengue haemorrhagic fever/ shock syndromes (DHF/DSS). It is estimated that at least 50 million dengue infections and 250000 cases of DHF occur annually. DHF is characterised by an increase in vascular permeability which is believed to be immune mediated and has been associated with elevated levels of inflammatory cytokines in the blood of DENV infected individuals. Our current understanding of dengue pathogenesis is limited and there are no vaccines or antiviral treatments in use to control the spread of DENV.
DENV non-structural protein 5 (NS5) plays a key role in virus replication, containing enzymatic activities required for replication of the DENV RNA genome. Recent studies have shown that the flavivirus NS5 protein may also play a role in viral pathogenesis. The NS5 proteins of a number of flaviviruses have been shown to inhibit interferon signalling. By contrast, the DENV NS5 protein has been shown to induce the production of interleukin-8 (IL-8), a cytokine that has been associated with severe DENV disease.
We hypothesise that the DENV NS5 protein interacts with components of the signalling pathways involved in the regulation of host cell genes, including IL-8, in DENV infected cells. The overall aim of this proposal, is to investigate how the DENV NS5 protein interacts with cellular signalling pathways, define the regions of NS5 involved and determine whether there are differences in the induction of IL-8 by the NS5 protein of different DENV strains. To achieve this aim the effects of expressing i) the NS5 gene from different DENV strains and ii) mutated versions of the NS5 gene on IL-8 gene transcription in cultured cells will be investigated. A monocytic cell line stably expressing NS5 will then be used to identify cellular proteins that interact with NS5 and determine how NS5 induces IL-8 gene transcription.
By understanding how individual DENV gene products modulate host cell processes, we will begin to elucidate the role of viral factors in DENV pathogenesis. This knowledge can be used to develop improved DENV vaccines and antiviral agents. In addition, the research proposed in this application has the potential to identify a novel host-pathogen interaction of general importance.

Publications

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Davidson AD (2009) Chapter 2. New insights into flavivirus nonstructural protein 5. in Advances in virus research

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Davidson AD (2017) Proteomics technique opens new frontiers in mobilome research. in Mobile genetic elements

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Dejnirattisai W (2011) Lectin switching during dengue virus infection. in The Journal of infectious diseases

 
Description MRC - Newton Fund UK-Philippines Joint Health Research Fund
Amount £200,000 (GBP)
Funding ID MR/N019245/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 12/2018
 
Description Mahidol University, Thailand PhD Scholarship
Amount £60,000 (GBP)
Organisation Mahidol University 
Sector Academic/University
Country Thailand
Start 10/2015 
End 09/2019
 
Description Newton MRC-NSTDA UK- Thai fund
Amount £714,015 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Royal Society International Exchange Scheme
Amount £8,700 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2014 
End 03/2016
 
Description Saudi government PhD scholarship
Amount £90,000 (GBP)
Organisation Government of Saudi Arabia 
Sector Public
Country Saudi Arabia
Start 10/2011 
End 10/2015
 
Description Saudi government PhD scholarship
Amount £90,000 (GBP)
Organisation Government of Saudi Arabia 
Sector Public
Country Saudi Arabia
Start 02/2012 
End 02/2016
 
Description Sir Henry Wellcome Postdoctoral Fellowship
Amount £250,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 03/2016
 
Title Proteomics informed by transcriptomics analysis of Aedes aegyptii Aag2 cells 
Description We used a novel proteomics informed by transcriptomics (PIT) approach to analyse Aedes aegyptii Aag2 cells. The proteins encoded by approximately 6,500 transcripts were identified, vastly increasing the number of experimentally confirmed Ae. aegypti proteins. The analysis provided 145 new genome annotations, the majority of which did not lie in either regions of poor sequence quality or mapping data, suggesting that the completeness of a genome's assembly may not be a major driver behind gaps in annotation. The utility of analysis for guiding annotation efforts was demonstrated by the identification of chromosome 1 and chromosomal loci 1p3, 1q4 and 2p4 as hotspots of poor annotation. We also detected 137 proteins expressed by transposable elements, representing the first proteomic characterisation of an organism's mobilome. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact none as yet 
URL http://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-016-3432-5
 
Description High-throughput interactomic analysis of the dengue virus NS5 protein. 
Organisation Mahidol University
Department Institute of Molecular Biosciences
Country Thailand 
Sector Academic/University 
PI Contribution The laboratory at the University of Bristol has developed stable cell lines expressing the dengue virus NS5 protein which are being used for high-throughput proteomic analysis.
Collaborator Contribution A Thai MSc exchange student worked in the University of Bristol laboratory for 3 months preparing samples for high-throughput analysis and learning how to analyse the data. The data is now being used by both laboratories for further collaborative studies on dengue virus replication.
Impact Joint grant application to the MRC UK-Thai Newton funding scheme which was successful. A larger project encompassing this collaboration will now start in April, 2018.
Start Year 2015
 
Description Interaction of the dengue virus NS5 protein with RNA structures 
Organisation University of Leeds
Department Faculty of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I have provided dengue virus genetic systems and plasmids encoding dengue virus proteins to the collaborator. i have also provided the collaborator with intellectual input that can be used to investigate host proteins involved in virus replication.
Collaborator Contribution Producing protein using the plasmid constructs, RNA interaction studies, proteins and RNA structural studies.
Impact Dynamic interactions between trans-activating factors and structured RNA during dengue virus replication. Lauren Branfield, Andrew Davidson, Mark Harris, Anastasia Zhuravleva and Andrew Tuplin Society of Microbiology Annual Conference, 2016 Dynamic interactions between trans-activating factors and structured RNA during dengue virus replication Lauren Branfield, Andrew Davidson, Chi Trinh, Mark Harris, Anastasia Zhuravleva and Andrew Tuplin Society of Microbiology Annual Conference, 2017
Start Year 2015
 
Description Project 3D-Targets: The UK-Philippines Dengue Diagnostic and Drug targets Consortium 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration involves an integrated omics analysis of clinical samples from patients in the Philippines with different grades of dengue disease severity in order to identify prognostic biomarkers. My research team is developing integrated computational analysis methods and using high throughput protemics to analyse clinical samples.
Collaborator Contribution The collaboration involves an integrated omics analysis of clinical samples from patients in the Philippines with different grades of dengue disease severity in order to identify prognostic biomarkers. The collaborators are developing integrated computational analysis methods and using high throughput metagenomics and transcriptomics to analyse clinical samples. UPM is collecting clinical samples and has produced a biobank.
Impact UK-Philippines Joint Health Research Project 3D-Targets: The UK-Philippines Dengue Diagnostic and Drug targets Consortium
Start Year 2015
 
Description Project 3D-Targets: The UK-Philippines Dengue Diagnostic and Drug targets Consortium 
Organisation University of Philippines Manila
Country Philippines 
Sector Academic/University 
PI Contribution The collaboration involves an integrated omics analysis of clinical samples from patients in the Philippines with different grades of dengue disease severity in order to identify prognostic biomarkers. My research team is developing integrated computational analysis methods and using high throughput protemics to analyse clinical samples.
Collaborator Contribution The collaboration involves an integrated omics analysis of clinical samples from patients in the Philippines with different grades of dengue disease severity in order to identify prognostic biomarkers. The collaborators are developing integrated computational analysis methods and using high throughput metagenomics and transcriptomics to analyse clinical samples. UPM is collecting clinical samples and has produced a biobank.
Impact UK-Philippines Joint Health Research Project 3D-Targets: The UK-Philippines Dengue Diagnostic and Drug targets Consortium
Start Year 2015
 
Description Structure function analysis of the dengue virus NS5 protein 
Organisation Novartis
Department Institute for Tropical Diseases Research
Country Singapore 
Sector Multiple 
PI Contribution We are investigating the effects of mutations in the viral NS5 protein using reverse genetics.
Collaborator Contribution The group at NITD is investigating the effects of mutations in the viral NS5 protein using recombinant protein based assays.
Impact Lim, S.P., Koh, J.H.K, Seh, C-C, Liew, C-W, Davidson, A.D., Chua, L.S., Chandrasekran, R., Cornvik, T., Shi, P-Y and Lescar, J. (2013). A crystal structure of the Dengue virus NS5 polymerase delineates inter-domain amino acids residues that enhance its thermostability and de novo initiation activities. Journal of Biological Chemistry 288: 31105-31114. First Published on September 11, 2013, doi:10.1074/jbc.M113.508606 The collaboration involves structural biologists and virologists.
Start Year 2012
 
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 RW talked to 50 school students about her research and a career in science.

N/A
Year(s) Of Engagement Activity 2008
 
Description UK-Philippines Infectious Diseases Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop to foster collaborative research between UK and Filipino scientists.
Year(s) Of Engagement Activity 2015
 
Description Visit to SE Asia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other audiences
Results and Impact 150 scientists from South-east Asia attended a workshop organised by the British High Commission to highlight UK research on dengue virus.

Europe-South East Asia Symposium on Dengue, 5-6 August, 2010. University of Malaya, Kuala Lumpur, Malaysia. http://tidrec.um.edu.my/dengue/
Dengue virus - host cell interactions: novel targets for antiviral strategies and diagnostics? Andrew Davidson

Location and year: University of Malaya, Malaysia and Mahidol University, Thailand, 2010


Established a collaborative scientific network to support an application to the EU FP7 programme.
Year(s) Of Engagement Activity 2010
URL http://tidrec.um.edu.my/dengue/