Regulatory role of non-coding RNAs in herpesvirus infections

Lead Research Organisation: University of Cambridge
Department Name: Medicine

Abstract

Herpesviruses are medically relevant causing a variety of diseases ranging from the common cold sore to cancer. During evolution these viruses have learned to usurp numerous cellular processes and counteract any host defence mechanism. Therefore, they provide ideal tools to study fundamental biological processes.

Recently, small molecules called microRNAs were identified as key regulators in multicellular organisms and some viruses, predominantly herpesviruses. Virally-encoded microRNAs may represent novel targets for highly specific antiviral drugs. Animal models are required to understand their function throughout the virus life-cycle. The human cytomegalovirus is the leading cause of birth defects resulting from congenital infections and causes severe disease in immunocompromised patients. My group established its murine model to study viral miRNA function in vivo. In close collaboration with a network of other national and international groups this project aims at elucidating the role of cytomegalovirus miRNAs and their application as targets for novel, highly specific antiviral drugs.
I developed a novel system based on metabolic labelling of nascent RNA which allows precise monitoring the kinetics of cellular and viral gene expression. In a second project I will combine this with next-generation sequencing and whole-proteome mass-spectrometry to study virus-host interaction with superior resolution.

Technical Summary

All herpesviruses share the ability to persist for life in infected individuals thus posing the risk of reactivation and disease. The eight human herpesviruses inflict a substantial burden of disease and economic costs in immunosuppressed patients and in pregnancy. Our lack of understanding of the molecular mechanisms governing viral gene expression and manipulation of host gene expression significantly hinders our ability to deal with the associated diseases and has obstructed the development of new therapies.

Here I propose two closely related projects which ask three major questions.
1) How do herpesviruses modulate cellular and viral gene expression?
2) What is the contribution and function of herpesvirus miRNAs?
3) Can viral miRNAs serve as targets for novel antiviral drugs?

The low temporal resolution of standard gene expression profiling and the inability to precisely measure RNA decay has severely hampered our understanding of the regulation of gene expression during virus infections. We developed a novel system for nascent transcriptome analysis (NTA) to overcome all of these problems. We now combine NTA with next-generation sequencing to study regulation of gene expression during lytic MCMV and HCMV infection. Similar approaches will be applied to the ?- and ?-herpesvirus family (HSV-1, MHV68). Thereby, we will provide a comprehensive picture of the regulation of gene expression in herpesvirus infection. SILAC proteomics available at the Babraham Institute will support NTA data at whole proteome level but will also elucidate the effects of viral miRNAs on target protein synthesis.

In close collaboration with a number of international groups my group established the infection of the mouse with the murine cytomegalovirus (MCMV) to study the function of cytomegalovirus miRNAs in vivo. Technologies like traceless-mutagenesis of viral genomes, miRNA target identification by RIP-Chip as well as in vivo experiments are well established. We will now study how viral miRNAs concert with viral proteins to provide a favourable environment for these viruses in vivo and how this knowledge can be used against them.

Viral miRNA research is a highly competitive field of research. Based on the advantage of a timely start and well established collaborations with leading groups in the field I am confident that both of these studies are straightforward. The aims are realistically achievable and the outcome will greatly improve our understanding of herpesvirus biology in general. Most importantly, the methodological approaches are easily transferred to any other cellular process opening up a plethora of opportunities for the future.

Publications

10 25 50
 
Description DFG project grant
Amount € 142,650 (EUR)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 10/2015 
End 09/2018
 
Description ERANET (Infect-ERA)
Amount € 324,000 (EUR)
Organisation EU-T0 
Sector Public
Country European Union (EU)
Start 06/2015 
End 05/2018
 
Description ERC Consolidator Grant
Amount € 2,000,000 (EUR)
Funding ID 721016 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 04/2017 
End 03/2022
 
Description Humboldt Postdoctoral Fellowship
Amount € 150,000 (EUR)
Organisation Alexander von Humboldt Foundation 
Sector Public
Country Germany
Start 10/2015 
End 09/2017
 
Description NHSBT Core Funding
Amount £438,000 (GBP)
Organisation NHS Blood and Transplant (NHSBT) 
Sector Public
Country United Kingdom
Start 04/2012 
End 10/2015
 
Title 4-thiouridine tagging of newly transcribed RNA 
Description A method to metabolically label and purify newly transcribed RNA using 4-thiouridine. This allows high-resolution gene expression profiling to study real-time RNA synthesis, processing and decay. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact In many fields of research, studies are limited by the poor temporal resolution of standard gene expression profiling using total cellular RNA, inadequate techniques to determine RNA half-lives and the lack of methods to study RNA processing. Our approach allows overcoming all of these limitations and is starting to be recognised as such in the field. 
 
Title Ribosome Profiling 
Description Real-time analysis of translational activity by purification, cloning and large-scale sequencing of ribosome-protected RNA fragments. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact First data on murine cytomegalovirus and herpes simplex virus translatome. 
 
Description High-Resolution Gene Expression Profiling 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution Develop 4-thiouridine (4sU)-tagging of nascent RNA. Project design and technical advice
Collaborator Contribution Applications of 4sU-tagging
Impact Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection. Marcinowski L et al., PLoS Pathog. 2012 Sep;8(9):e1002908. Epub 2012 Sep 6. Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager et al., Genome Res. 2012 Apr 26. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Miller C et al., Mol Syst Biol. 2011 Jan 4;7:458. The phosphoproteome of toll-like receptor-activated macrophages. Weintz G et al., Mol Syst Biol. 2010 Jun 8;6:371. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC et al., Nucleic Acids Res. 2009 Sep;37(17):e115. Epub 2009 Jun 26.
Start Year 2010
 
Description High-Resolution Gene Expression Profiling 
Organisation Braunschweig University of Technology
Department Helmholtz-Zentrum für Infektionsforschung
Country Germany 
Sector Academic/University 
PI Contribution Develop 4-thiouridine (4sU)-tagging of nascent RNA. Project design and technical advice
Collaborator Contribution Applications of 4sU-tagging
Impact Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection. Marcinowski L et al., PLoS Pathog. 2012 Sep;8(9):e1002908. Epub 2012 Sep 6. Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager et al., Genome Res. 2012 Apr 26. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Miller C et al., Mol Syst Biol. 2011 Jan 4;7:458. The phosphoproteome of toll-like receptor-activated macrophages. Weintz G et al., Mol Syst Biol. 2010 Jun 8;6:371. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC et al., Nucleic Acids Res. 2009 Sep;37(17):e115. Epub 2009 Jun 26.
Start Year 2010
 
Description High-Resolution Gene Expression Profiling 
Organisation Heinrich Heine University Düsseldorf
Department Institute of Virology
Country Germany 
Sector Academic/University 
PI Contribution Develop 4-thiouridine (4sU)-tagging of nascent RNA. Project design and technical advice
Collaborator Contribution Applications of 4sU-tagging
Impact Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection. Marcinowski L et al., PLoS Pathog. 2012 Sep;8(9):e1002908. Epub 2012 Sep 6. Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager et al., Genome Res. 2012 Apr 26. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Miller C et al., Mol Syst Biol. 2011 Jan 4;7:458. The phosphoproteome of toll-like receptor-activated macrophages. Weintz G et al., Mol Syst Biol. 2010 Jun 8;6:371. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC et al., Nucleic Acids Res. 2009 Sep;37(17):e115. Epub 2009 Jun 26.
Start Year 2010
 
Description High-Resolution Gene Expression Profiling 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Gene Center Munich
Country Germany 
Sector Academic/University 
PI Contribution Develop 4-thiouridine (4sU)-tagging of nascent RNA. Project design and technical advice
Collaborator Contribution Applications of 4sU-tagging
Impact Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection. Marcinowski L et al., PLoS Pathog. 2012 Sep;8(9):e1002908. Epub 2012 Sep 6. Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager et al., Genome Res. 2012 Apr 26. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Miller C et al., Mol Syst Biol. 2011 Jan 4;7:458. The phosphoproteome of toll-like receptor-activated macrophages. Weintz G et al., Mol Syst Biol. 2010 Jun 8;6:371. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC et al., Nucleic Acids Res. 2009 Sep;37(17):e115. Epub 2009 Jun 26.
Start Year 2010
 
Description High-Resolution Gene Expression Profiling 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Institute for Informatics
Country Germany 
Sector Academic/University 
PI Contribution Develop 4-thiouridine (4sU)-tagging of nascent RNA. Project design and technical advice
Collaborator Contribution Applications of 4sU-tagging
Impact Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection. Marcinowski L et al., PLoS Pathog. 2012 Sep;8(9):e1002908. Epub 2012 Sep 6. Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager et al., Genome Res. 2012 Apr 26. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Miller C et al., Mol Syst Biol. 2011 Jan 4;7:458. The phosphoproteome of toll-like receptor-activated macrophages. Weintz G et al., Mol Syst Biol. 2010 Jun 8;6:371. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC et al., Nucleic Acids Res. 2009 Sep;37(17):e115. Epub 2009 Jun 26.
Start Year 2010
 
Description MicroRNAs in Herpesvirus Infections 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Gene Center Munich
Country Germany 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Institute for Informatics
Country Germany 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Department Max von Pettenkofer-Institute of Hygiene and Medical Microbiology
Country Germany 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation University of Edinburgh
Department Division of Pathway Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation University of Regensburg
Department Institute for Biophysics & Physical Biochemistry
Country Germany 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007
 
Description MicroRNAs in Herpesvirus Infections 
Organisation University of Strasbourg
Country France 
Sector Academic/University 
PI Contribution Establish and provide cutting-edge high-throughput technologies including RIP-Chip, PAR-CLIP and 4sU-tagging to the consortium.
Collaborator Contribution Know-how Models Target validation and functional assays Antibodies Bioinformatic analysis
Impact Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. Marcinowski L et al., PLoS Pathog. 2012 Feb;8(2):e1002510. Epub 2012 Feb 9 Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B) Tuddenham L et al., J Virol. 2012 Feb; 86(3):1638-49. Epub 2011 Nov 23 Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands. Dölken L et al., PLoS Pathog. 2010 Oct 14;6(10):e1001150. Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L et al., Cell Host Microbe. 2010 Apr 22;7(4):324-34.
Start Year 2007