Probing the translational dynamics of influenza virus infection.

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

At the cellular level, the outcome of virus infection hinges on a battle between virus and cell for the control of the cellular biosynthetic machinery, as this ultimately determines the amount of progeny virus produced. Many viruses encode their own nucleic acid polymerases, but all are reliant on the cellular protein synthesis machinery. The ribosome therefore represents the key battleground. Using the major human and animal pathogen influenza A virus (IAV) as a model system, we will use the novel technique of ribosomal profiling to detail this conflict at a level of detail hitherto impossible. Ribosomal profiling allows the accurate determination of which mRNAs (viral or cellular) that the ribosomes are translating at a given moment; thereby defining the state of the 'frontline' in the battle for control of the cell. Our objectives are to:

1. Define the cellular and viral mRNAs that are translated in virus-host cell combinations where either the virus 'wins' or where the host wins.

2. To define which of the many viral mechanisms that affect cellular gene expression are and their specific targets which decide the outcome of the battle for control of the cellular translation machinery.

3. Test the hypothesis that IAV specifically manipulates the ribosome to bias it towards preferential translation of viral messengers.


Overall, the project will define the key events from both virus and cellular perspective that define whether an individual infected cell is productively infected; data that will aid the design of new therapeutic intervention strategies.

Technical Summary

In order to produce progeny virions, the major human and animal pathogen influenza A virus (IAV) uses the host cell translational machinery to synthesize viral proteins. Simultaneously, the virus attempts to inhibit cellular macromolecular synthesis; both to free up the translation machinery and to block the synthesis of antiviral response proteins. IAV has evolved several seemingly redundant shutoff mechanisms, highlighting the importance of this aspect of virus infection. However, whilst this reinforces this subject as an area for potential therapeutic intervention, the key molecular events from either virus or host perspective are obscure.

Our objectives are to:

1. Utilise the recently developed technique of ribosomal profiling in conjunction with RNAseq to define the cellular and viral 'translatome' in permissive (293T and A549 cells) and non-permissive (monocyte-derived macrophages) cell types and with human-adapted (A/PR/8/34, A/Udorn/72) and avian (A/mallard/NL/10/99) strains of virus.

2. To define the key viral mechanisms and cellular targets that decide the outcome of the battle for control of the cellular translation machinery, by comparing the translatomes of wild type and mutant strains of IAV disabled for specific shut-off functions; PA-X mutants, PB2/PA mutants defective in RNA Pol II degradation and various NS1 mutants defective for CPSF, PAF1, eIF4GI binding.

3. Test the hypothesis that IAV specifically manipulates the biogenesis and/or composition of ribosomes to bias them towards viral translation, using an unbiased affinity purification method followed by mass spectrometry as well as testing specific hypotheses involving viral interactions with the NOH61 helicase and the PELP1, LAS1L, TEX10 and WDR18 complex.


The project will define the key events from both virus and cellular perspective that define the output of progeny virions from an individual infected cell; data that will aid of new therapeutic intervention strategies.

Planned Impact

1. A deeper understanding of influenza virus.
IAV is the etiologic agent of influenza, one of the most economically significant diseases of man. The virus causes great morbidity, substantial mortality and has the potential to spread in devastating pandemics. Rapid evolution of IAV persistently complicates the effectiveness of vaccines and therapeutics; presently, there is no effective treatment, and repeated vaccination is necessary against newly circulating strains. A better understanding of the complex host cell pathways co-opted by influenza virus for replication, the means by which the virus gains control and (conversely), the ways in which the cell successfully defends itself, will provide new targets and strategies for antiviral therapy. This information will also be of interest to the biomedical industry and to the general public; IAV is an economically attractive target for the former, and an endless source of interest to the latter.

2. Ribosomal profiling - a new tool in the armoury
Presently, our knowledge of how cells respond to infection, and how the virus counteracts this, is incomplete, particularly at the level of protein synthesis. Furthermore, we know little of how IAV interacts with the host translation apparatus, nor how the cell's translatome is modulated by the many host cell shut off strategies the virus can employ. Ribosomal profiling can provide the ability to rapidly identify specific host factors involved in these pathways, allowing for novel disease intervention strategies. It is a relatively new technique but being increasingly used in the study of translation in healthy and disease states. The progress we have made so far in optimising the system - and the insights will undoubtedly gain during the course of this work - together with the bioinformatics pipeline in development with our collaborator Andrew Firth, leads us to believe that our findings will be of broad use to many others in the field of translation and beyond.

3. Ribosomal profiling - insights into gene expression
Based on published work and our own preliminary work, it is highly likely that the ribosomal profiling studies will identify novel ribosomal pausing signals and unusual examples of translational control and even (potentially) novel genes. All of this information will increase our understanding of the diversity of gene expression in mammalian cells and virus-infected cells. This information will be of wide scientific interest and may have implications in biotechnology and disease.

4. Training and career development.
The grant will provide significant benefit to the careers of the junior staff involved. It will enable Drs Wise and Irigoyen to develop their independent careers, as well as training the junior PDRA at the RI in a wide range of scientific and transferable skills that will benefit their future development. We also anticipate that our internal presentations of the research within our host Institutions will provoke interest in a new technology that will lead to our training other researchers in the technique.
 
Description Wellcome Investigator Award
Amount £774,356 (GBP)
Funding ID 202797/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 10/2021
 
Title DSN profiling 
Description We have adapted the use of duplex-specific nuclease to minimise ribosomal RNA contamination in ribosome profiling datasets. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Other laboratories have now adapted this technique to carry out sequence-independent rRNA removal from profiling libraries. 
 
Title Ribosomal profiling data from murine leukemia virus infected cells 
Description Ribosome profiling and RNA sequencing dataset from Rat 2 cells infected with the retrovirus murine leukemia virus. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact This is the first ribosomal frameshifting dataset for any retrovirus. The datasets generated and analysed during the current study have been deposited in the EBI ArrayExpress database under the accession number E-MTAB-6231. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6231/
 
Title Ribosome profiling data from DSN paper 
Description Raw and processed data files from ribosome profiling of mouse cells 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This high-resolution dataset has been downloaded extensively. 
URL http://www.ebi.ac.uk/arrayexpress
 
Title Ribosome profiling data from mouse hepatitis coronavirus infected cells 
Description Ribosome profiling datasets from virus infected mouse cells. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact This dataset was the first published analysis of RNA virus infection through ribosome profiling 
URL http://www.ebi.ac.uk/arrayexpress
 
Description Flu collaboration 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration is to carry out ribosome profiling of influenza infection. Professor Digard's lab is preparing various flu recombinants, our role is to look at virus and host translation in cells infected by these flu variants.
Collaborator Contribution The partner provides viruses and antibodies.
Impact Development of the ribosome profiling methodology has led to two publications so far. We are currently analysing the flu data.
Start Year 2015
 
Description Flu translation 
Organisation University of Glasgow
Department Institute of Infection, Immunity and Inflammation
Country United Kingdom 
Sector Academic/University 
PI Contribution Our analysis of ribosomal frameshifting in influenza infected cells generated a dataset of sites of initiation of translation in influenza segment mRNAs and alternative initiation sites in the short cap-snatched segments of cellular mRNAs that are attached to the 5' end of all flu segments. This dataset was shared with Dr Edward Hutchinson at the University of Glasgow who had a hypothesis that viral protein diversity in influenza virus might be achieved through alternative initiation of translation in the cellular mRNA stretches of flu mRNAs. This collaboration has lead to the submission of a manuscript on this topic to Cell and may form the basis of a future grant application.
Collaborator Contribution Dr Hutchinson provided a range of experimental datasets including mass spectroscopy of flu proteins.
Impact This mutlidisciplinary collaboration has established upstream translation initiation as a novel mechanism of influenza gene expression. The work is currently submitted to Cell and a preprint has been placed on the biorxiv server (address above).
Start Year 2019
 
Title riboSeqR software 
Description Software for plotting functions, frameshift detection and parsing of sequencing data from ribosome profiling experiments. It was developed by Thomas Hardcastle for analysis of ribosomal profiling data in Chung et al. DSN paper (d.o.i. below). 
Type Of Technology Software 
Year Produced 2015 
Open Source License? Yes  
Impact Some 5000 downloads since first uploaded. 
URL http://bioconductor.org/packages/release/bioc/html/riboSeqR.html
 
Description ASM Biodefense and Emerging Diseases Meeting, Washington DC, February 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk on Coronavirus replication given to attendees of the 2015 Biodefense Meeting. The aim was to give a broad audience a background to how ribosome profiling can be used to find out information about virus replication and host-virus interactions. This was particularly relevant given the recent emergence of novel disease-associated coronaviruses like SARS and MERS.
Year(s) Of Engagement Activity 2015
URL https://www.asm.org/index.php/choma/71-membership/archives/834-biodefense-meetings-historical-list
 
Description Ribosomal Profiling 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 I co-organised a Biochemical Society Workshop on Ribosome Profiling (https://www.biochemistry.org/Events/tabid/379/MeetingNo/WS020/view/Conference/Default.aspx). This was a whole day dedicated to the methodology of this technique, including wet-lab work and bioinformatics. It was presented to a very diverse audience of 50-100 individuals with a focus on problem solving. It generated a great deal of interest, discussion and feedback.
Year(s) Of Engagement Activity 2016
URL http://data.plantsci.cam.ac.uk/ribosome-footprinting/