CE15 - Immunologie, Infectiologie et Inflammation

Control of type A Influenza virus Propagation and Immune Responses: Investigating the role of NCOA7-Alternative Start – CAIPIRINAS

Role and mechanism of action of NCOA7-AS antiviral protein in vitro and in vivo

Influenza viruses are major pathogenic agents. Current strategies to circumvent influenza infection involve the vaccination against seasonal viruses and few antiviral drugs, with limited efficiency. A better understanding of influenza A virus propagation in infected hosts and its control by the immune system is therefore essential to develop future therapeutic strategies. In this context, CAPIRINAS proposes to study in depth the role and mechanism of action of the antiviral protein NCOA7-AS.

Control of type A Influenza virus Propagation and Immune Responses: Investigating the role of NCOA7-Alternative Start

Influenza A virus (IAV) enters the cells via endocytosis and a pH decrease is required for viral and endosomal membranes fusion, and subsequent access of the viral ribonucleoprotein complexes to the cytosol. Endosomal acidification is key to antigen degradation and presentation to immune cells. We have recently identified the short isoform of NCOA7 (NCOA7-AS, for Alternative Start) as an interferon (IFN)-induced protein that limits IAV entry. NCOA7-AS interacts with the vacuolar (V-)ATPase, the proton pump responsible for endosomal acidification. Through an as yet unelucidated mechanism, this interaction leads to increased endosomal acidification and antigen degradation, and is detrimental to IAV entry into target cells. <br />To understand how NCOA7-AS regulates the V-ATPase activity, we have performed a mass spectrometry analysis to identify its cellular binding partners and we now propose to explore their role in NCOA7-AS’s activity. Moreover, we propose to conduct a thorough structure-function analysis of NCOA7-AS to gain insights into its molecular mechanisms of action. In parallel, we have generated mice deficient for NCOA7-AS using CRISPR Cas9, to assess NCOA7-AS’s role following AIV infection, in vivo. Finally, we hypothesize that NCOA7-AS may not only be important in the innate immune (IFN-regulated) responses against IAV, but may also play a role in the downstream adaptive immune responses (through a role for NCOA7-AS in antigen presentation and possibly in CD8 cytotoxicity, that relies on acidification of their lytic granules). <br />In this context, the objectives of CAIPIRINAS (Control of type A Influenza virus Propagation and Immune Responses: Investigating the role of NCOA7-Alternative Start) are the following: <br />Aim 1- Deciphering the detailed molecular mechanism(s) of action of NCOA7-AS <br />Aim 2- Elucidating NCOA7-AS’s role in the control of IAV in vivo and in the establishment of the immune responses against this virus.

Structural biology methods, biochemistry, cell biology, molecular biology, CRISPR, classical methods for studying viral replication in vivo and in vitro, methods for studying immune responses in vivo

We have validated the interaction between NCOA7-AS and the most interesting binding-partners identified, which are involved in vesicular trafficking but have interestingly never been linked to the V-ATPase activity. We have been able to solve the three-dimensional structure of the TLDc domain of NCOA7-AS, as well as the structure of a domain of NCOA7-AS in complex with an important partner. We have generated NCOA7-AS knockout mice and we are currently studying the impact of NCOA7-AS knockout on IAV replication and on immune responses.

This project will shed light on the mechanism of action and role in vivo of a potent antiviral protein, NCOA7-AS.

Arnaud-Arnould M., Tauziet M., Moncorgé O., Goujon C., Blaise M. Crystal structure of the TLDc domain of human NCOA7-AS. Acta Crystallographica Section F: Structural Biology Communications, 2021, 77(8).

McKellar J., Rebendenne A., Wencker M., Moncorgé O., Goujon C. Mammalian and Avian Host Cell Influenza A Restriction Factors. Viruses, 2021, 13(3):522. (review)

Influenza A and B viruses (IAV and IBV) are major pathogenic agents, causing around 500 000 deaths yearly. The possibility of a new IAV pandemic was ranked in the top 10 threats for human health in 2019 by the World Health Organization. This threat particularly resonates in the context of the current COVID-19 pandemic, which shows the consequences of the global spread of a dangerous respiratory virus on health care systems, on the economy and more globally on our societies.
Current strategies to circumvent influenza infection involve the vaccination against seasonal viruses and two approved classes of antiviral drugs in Europe, with major resistance problems and limited efficiency. In the case of a new IAV pandemics, development and large-scale production of a vaccine would require at least 8 months. A better understanding of IAV propagation in infected hosts /nd its control by the immune system is therefore essential to develop future therapeutic strategies.
IAV enters the cells via endocytosis and a pH decrease is required for viral and endosomal membranes fusion, and subsequent access of the viral ribonucleoprotein complexes to the cytosol. Endosomal acidification is key to antigen degradation and presentation to immune cells. We have recently identified the short isoform of NCOA7 (NCOA7-AS, for Alternative Start) as an interferon (IFN)-induced protein that limits IAV entry. NCOA7-AS interacts with the vacuolar (V-)ATPase, the proton pump responsible for endosomal acidification. Through an as yet unelucidated mechanism, this interaction leads to increased endosomal acidification and antigen degradation, and is detrimental to IAV entry into target cells.
To understand how NCOA7-AS regulates the V-ATPase activity, we have performed a mass spectrometry analysis to identify its cellular binding partners and we now propose to explore their role in NCOA7-AS’s activity. Moreover, we propose to conduct a thorough structure-function analysis of NCOA7-AS to gain insights into its molecular mechanisms of action. In parallel, we have generated mice deficient for NCOA7-AS using CRISPR Cas9, to assess NCOA7-AS’s role following AIV infection, in vivo. Finally, we hypothesize that NCOA7-AS may not only be important in the innate immune (IFN-regulated) responses against IAV, but may also play a role in the downstream adaptive immune responses (through a role for NCOA7-AS in antigen presentation and possibly in CD8 cytotoxicity, that relies on acidification of their lytic granules).
In this context, the objectives of CAIPIRINAS (Control of type A Influenza virus Propagation and Immune Responses: Investigating the role of NCOA7-Alternative Start) are the following:
Aim 1- Deciphering the detailed molecular mechanism(s) of action of NCOA7-AS
Aim 2- Elucidating NCOA7-AS’s role in the control of IAV in vivo and in the establishment of the immune responses against this virus.
We have validated the interaction between NCOA7-AS and the most interesting binding-partners identified, which are involved in vesicular trafficking but have interestingly never been linked to the V-ATPase activity. We have been able to obtain crystals of recombinant NCOA7-AS, which will be used to solve, for the first time, the structure of this protein. Finally, we have generated NCOA7-AS knockout mice. Altogether our project is based on solid preliminary data and we have all the tools to reach our goals.
To sum up, CAIPIRINAS is a unique transdisciplinary project offering to study in depth an antiviral protein from its three-dimensional structure, its impact on the physiology of the cell and on viral infection, to its role in the immune control of IAV infection in vivo, in a mouse model. This project will undoubtedly shed light not only into the natural defense mechanisms against viral infections, on the links between innate and adaptive immunity, but also on the regulation of a protein complex involved in myriad cellular processes and many diseases, the V-ATPase.

Project coordinator

Madame Caroline GOUJON (Institut de Recherche en Infectiologie de Montpellier)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CIRI CENTRE INTERNATIONAL DE RECHERCHE EN INFECTIOLOGIE
IRIM Institut de Recherche en Infectiologie de Montpellier
IRIM Institut de Recherche en Infectiologie de Montpellier

Help of the ANR 443,712 euros
Beginning and duration of the scientific project: December 2020 - 42 Months

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