Controlling Influenza A Virus Liquid Organelles

Project Information

LOFlu

Grant agreement ID: 101001521

DOI

10.3030/101001521

EC signature date 1 February 2021

Start date 1 April 2021

End date 31 July 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 870 250,00

EU contribution

€ 2 870 250,00

Coordinated by

UNIVERSIDADE CATOLICA PORTUGUESA
Portugal

Periodic Reporting for period 3 - LOFlu (Controlling Influenza A Virus Liquid Organelles)

Reporting period: 2024-04-01 to 2024-08-31

Summary of the context and overall objectives of the project

The World Health Organization monitors viral infections worldwide with the aim of coordinating strategies to control viral outbreaks. The on-demand development of vaccines or antibody treatment does not confer a first line of defense against unpredictable infections caused by new viruses in humans such as pandemic influenza, corona or Ebola viruses, and new approaches are needed. We propose to investigate the fundamental basis of novel host-pathogen interactions in influenza A virus (IAV) infection that may define new antiviral strategies. We discovered that the important pathogen IAV induces the intracellular assembly of viral inclusions that behave like liquid organelles. IAV inclusions serve as assembly sites for the IAV segmented genome, a key step in the viral lifecycle. We now find that the maintenance of the liquid character of IAV inclusions is essential for viral replication. As we identified some of the host and viral components of IAV inclusions, we now have the tools to interrogate how specific interactions and cellular processes result in phase-separated compartments. We aim to learn how the function of IAV inclusions is related to their material state and investigate the potential of imposing phase transitions in an organism to limit IAV infection. Phase separation provides a novel conceptual framework to tackle how viruses exploit cells to organize viral reactions in space and in time. It also provides alternative principles for exploring aspects of the IAV lifecycle not yet fully understood, including how influenza epidemic and pandemic genomes assemble. Taken together, we propose a new, integrated approach for studying phase separated phenomena, from the molecular to the organismal level, that will bring a deeper understanding and control to viral infections. Our work will also be of relevance to other fields of biomedicine, including in the science of soft matter which is involved in neurodegenerative diseases and some cancers.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Until now we developed experimental systems/tools to use in the LOFlu project and have make considerable scientific advances in all aims, as follows:

Aim 1: We identified components in influenza A virus liquid inclusions using a mass spectrometry-based approach called solubility proteome profiling. With this approach, we were able to identify how the cellular soluble changes during infection, and how these changes relate to the formation of viral inclusions. Now, we will validate the findings and investigate novel avenues that resulted from the screen. During the period covered by this report, my laboratory published new aspects of the mechanisms regulating the formation of influenza A virus liquid inclusions (Vale-Costa et al., PLoS Biology, 2023) and the remodeling of endosomal membranes into tubules (Jani et al., JCB, 2022), both relevant for the first aim of this project.

Aim 2: Tasks in Aim 2 are providing novel insights into the rules of hardening IAV liquid inclusions, which has been published in eLife (Etibor et al., 2023). For this work, we screened and compared how several different strategies for hardening influenza A virus liquid inclusions, ranked them and used the most efficient to further assess if it blocked viral infection in vitro and in vivo. We found that increasing the strength of interactions amongst progeny RNA hardened influenza A virus inclusions efficiently, but not increasing their concentration or the temperature of the system. This knowledge is essential for the rational design of drugs targeting the material properties of influenza A virus liquid inclusions. Moreover, we published our tools to quantify alterations in the material properties of influenza A virus inclusions (Etibor, O'riain et al., IJMS, 2023), and are in a unique position to perform screens identifying compounds and regulatory mechanisms not only altering the material properties but also rendering infection less efficient.

Aim 3: Major achievements include evaluation of viral inclusions in vivo. Using specific tools, we validated the phenotype of hardening viral inclusions in vivo because this is a very extreme morphological alteration of influenza A virus inclusions. In addition, with the solubility proteome profiling of Aim 1, we identified many changes in solubility in the cell upon infection that could provide alternative relevant functions to influenza A virus liquid inclusions. This is a very interesting new line of research within the grant. We implemented an in vitro reconstitution assay that will enable us to elucidate in vitro how influenza A genomes assemble.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

LOFlu is concentrating on the fundamental problem of compartmentalizing viral reactions. We build on our finding that some viral reactions during influenza A virus replication compartmentalize in molecular condensates also called viral inclusions that display liquid properties.
In LOFlu, we have started to elucidate the molecular underpinnings the formation and influenza A liquid inclusions. We are expanding our efforts to elucidate how the material properties are maintained and regulated in the cell. We have already shown that it is possible to harden viral inclusions and have defined the best strategies. This work supports the development of antivirals targeting the material properties of biomolecular condensates in viral infections. It also provides a framework for the selection of compounds with this activity for general application and thus provides an advance in disease therapy. We will now concentrate on the function of influenza A virus liquid inclusions during infection. Finally, we have identified which viral and host components undergo phase transitions during infection and are now understanding how phase transitions regulate host cell function and response to infection whilst facilitating or inhibiting viral replication. Such understanding will shed new light on how phase transitions operate as a new regulatory layer in the cell.

Hardening influenza A virus liquid inclusions renders infection less efficient