MRC Transition Support Award CDA Edward Hutchinson

This proposal is for Transition Support to continue the work of a Career Development Award (CDA), which was delayed and redirected in response to a number of unexpected issues. Both the CDA and the current proposal focus on influenza viruses. These cause seasonal influenza, which kills 290 000 - 650 000 people globally each year and is one of the leading global causes of death. As well as causing seasonal illnesses in humans each year, influenza viruses are unusually good at jumping from one species to another, as human and animal influenza viruses can exchange genes if they both infect the same host at the same time. The ready ability of influenza viruses to undergo this 'reassortment,' along with their high mutation rates, enables them to cause repeated and sometimes devastating pandemics.

The work in the original CDA focused on proteins in the virus particles that transmit influenza infections. We showed that these were much more variable than was previously appreciated. The host cell and the virus can both strongly influence which proteins are included in virus particles and in what amount, and the proteins themselves can be modified in different ways. We also discovered an entirely new class of 'hidden' influenza virus proteins, and examined how the virus particles produced in natural influenza virus infections can vary in shape, from the spherical particles typically studied in the laboratory to enormously extended filaments. As our work developed, we were able to examine more sources of variation among influenza virus particles: in the genes they carry, in the proteins that make them infectious and the in shapes they adopt.

This Transition Support proposal aims to consolidate this work. Work will begin by completing three well-advanced projects that were delayed during the CDA. The first of these draws together multiple lines of evidence to provide a picture of influenza virus particles in unprecedented detail, and showing where these microscopic structures can vary. The second shows how over time an infected cell alters the composition of the virus particles it sheds, making them more infectious just as the surrounding cells start to increase their antiviral defences. The third examines a set of chemical modifications to influenza virus proteins that can act like a series of switches, altering and regulating the proteins' functions.

With this work, we will have completed a wide-ranging survey of the ways in which influenza virus particles can vary, and developed many novel tools to study this variation. We will then ask how knowing about the enormous scope for variation among influenza virus particles changes our understanding of what happens when we get infected with influenza. The ability of influenza viruses to swap genes inside their hosts shows us that virus particles can interact during an infection, but most of our understanding of how influenza viruses actually work focusses on understanding the initiation of an infection by one single virus particle. Once an infection is underway in a host, we know that large numbers of highly variable virus particles are shed into a small space and can interact, but we have not yet been able to examine how interactions within this 'viral microenvironment' shape the outcome of an infection. Using the tools we have developed, we will examine for the first time how the single influenza virus particle that infects us, rapidly creates within us a swarm of highly variable virus particles that interact to determine the course of our infection. As well as detailing the course of a normal infection, this will allow us to understand how the 'swarms' created by a human and animal virus could come together to seed the next respiratory virus pandemic.

This Transition Support award aims firstly to complete three well-established studies, and then to consolidate those with the other findings of my CDA to enable a new way of understanding how influenza virus infections propagate within the host. The three studies are:

(1) A detailed, integrative model of the structure and composition of an influenza virion, combining proteomics, lipidomics, direct RNA sequencing and cryoelectron tomography to identify its fixed and flexible features.
(2) A novel mechanism whereby, as an infection proceeds, the virions shed from an infected cell change their composition and become more infectious, allowing them to overcome the antiviral defences of surrounding cells.
(3) Mapping the competing post-translational modifications of lysines in influenza virus proteins and identifying their regulatory effects.

Along with the work already completed in the CDA, these provide tools to examine how viruses propagate within a host. It is important to note that the host has typically been treated as a black box: initiation of infection by a single virus particle is extremely well-studied, as is transmission of virus particles between hosts. What happens within the host has been much harder to assess. We know that influenza viruses produce discrete lesions in the respiratory epithelium and replicate in them to high titres. We can infer from genome reassortment that coinfection within these lesions is common. And we have recently become aware that, although only a minority of influenza virus particles are fully infectious, a large proportion of particles are 'semi-infectious' and can drive an infection through complementation. By combining the tools we developed for studying variation in influenza virions with advanced experimental models of differentiated airway epithelia and with tracheal explants, we will provide the first experimental model of the heterogeneous collective of virions underpinning every natural influenza virus infection.