Periodic Reporting for period 1 - LOFlu (Controlling Influenza A Virus Liquid Organelles)
Periodo di rendicontazione: 2021-04-01 al 2022-08-31
The world health organization monitors viral infections worldwide with the aim to coordinate 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 that is involved in neurodegenerative diseases and some cancers.
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 that is involved in neurodegenerative diseases and some cancers.
Until now most of the research was dedicated to developing experimental systems/tools to use in the various aim, as follows:
Aim 1: We identified components in IAV 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. In addition, we will try to further identify the components within IAV liquid inclusions. For Aim 1.2 we already identified host processes essential for concentrating and forming IAV inclusions. In collaboration with Dr Petr Chlanda we are finalizing work showing how progeny RNA, host cell membranes and host cell factors cooperate to form liquid viral inclusions, at an ultrastructural level.
Aim 2: Tasks in Aim 2 are providing novel insights into the rules of hardening IAV liquid inclusions, which has been published in bioRxiv (Etibor et al., 2022). For this work, we screened and compared how several different putative strategies hardened IAV liquid inclusions, ranked them and used the most efficient to further assess that it blocked viral infection in vitro and in vivo. This knowledge is essential for the rational design of drugs targeting the material properties of IAV liquid inclusions. Using the knowledge acquired we have written a review (Etibor et al., 2021) and, with our developed tools we are in a unique position to start identifying cellular regulators able to manipulate the properties of IAV inclusions.
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 IAV inclusions. In addition, with the solubility proteome profiling of Aim 1, we identified that the viral protein non-structural protein 1 is insoluble during infection, which opens a new avenue to explore phase transitions during infection related to immunity (Aim 3.1). This is a very interesting new line of research within the grant. As soon as the in vitro reconstitution assays have been implemented, we will initiate aim 3.2 however in aim 1, we are evaluating the functional relevance of IAV inclusions using cells very successfully.
Aim 1: We identified components in IAV 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. In addition, we will try to further identify the components within IAV liquid inclusions. For Aim 1.2 we already identified host processes essential for concentrating and forming IAV inclusions. In collaboration with Dr Petr Chlanda we are finalizing work showing how progeny RNA, host cell membranes and host cell factors cooperate to form liquid viral inclusions, at an ultrastructural level.
Aim 2: Tasks in Aim 2 are providing novel insights into the rules of hardening IAV liquid inclusions, which has been published in bioRxiv (Etibor et al., 2022). For this work, we screened and compared how several different putative strategies hardened IAV liquid inclusions, ranked them and used the most efficient to further assess that it blocked viral infection in vitro and in vivo. This knowledge is essential for the rational design of drugs targeting the material properties of IAV liquid inclusions. Using the knowledge acquired we have written a review (Etibor et al., 2021) and, with our developed tools we are in a unique position to start identifying cellular regulators able to manipulate the properties of IAV inclusions.
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 IAV inclusions. In addition, with the solubility proteome profiling of Aim 1, we identified that the viral protein non-structural protein 1 is insoluble during infection, which opens a new avenue to explore phase transitions during infection related to immunity (Aim 3.1). This is a very interesting new line of research within the grant. As soon as the in vitro reconstitution assays have been implemented, we will initiate aim 3.2 however in aim 1, we are evaluating the functional relevance of IAV inclusions using cells very successfully.
LOFlu is concentrating on the fundamental problem of compartmentalizing viral reactions. We build on our finding that some viral reactions compartmentalize in molecular condensates also called viral inclusions that display liquid properties.
In LOFlu, we have started to elucidate the molecular underpinnings on 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 mechanisms that may modulate targeting the molecular interactions established within viral inclusions to provide novel antiviral strategies. 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.
In LOFlu, we have started to elucidate the molecular underpinnings on 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 mechanisms that may modulate targeting the molecular interactions established within viral inclusions to provide novel antiviral strategies. 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.