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.
