Viral RNA enriched exosomes are mediators of the interferon response – EXAMIN

To better our understanding of this newly discovered pathway of innate response activation, we analyze its regulatory mechanisms in the context of infection with two highly pathogenic human viruses for which infected cells similarly trigger robust IFN responses by pDCs. Especially, in the EXAMIN proposal, using multidisciplinary approaches, we plan to pursue the following aims:
I. Defining the biogenesis of PAMP-exosomes by HCV infected cells and their transmission to the pDCs. We will specifically determine:
1/ The ANXA2 function required for the biogenesis, trafficking and/or release of pDC activating signal
2/ The interplay between NS5A and ANXA2 in this process
3/ How HCV PAMP-exosomes are transmitted and captured by the pDCs
II. Defining the molecular basis of pDC activation by DENV infected cells. We will specifically elucidate:
1/ The modality of this short-range pDC activation: contribution of the exosomal pathway, viral determinant(s), phenotypic analysis of activated pDC and impact of infectious virus.
2/ The cellular factors specifically required within DENV infected cells.

DENV is the leading cause of mosquito-borne viral illness and death in humans. Several in vivo studies have demonstrated a key role of the innate immune response in the pathogenesis of DENV infection. We recently uncovered that sensing of DENV infected cells by the pDCs, triggers a robust Toll-like receptor (TLR)7-dependent IFN response. This sensing, which bypasses the blockage of IFN response in infected cells, induces additional antiviral features, including inflammatory cytokine secretion and pDC maturation. We demonstrated that pDC activation depends on cell-to-cell contact, a feature observed for West Nile virus, another member of the Flavivirus genus. We showed that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response. Finally, this concept might have broad importance for the many viruses that, like DENV, can prevent the pathogen-sensing machinery within infected cells and can release uncleaved glycoprotein-containing non-infectious particles.

By deciphering shared and specific host-virus interplays using evolutionary divergent viruses, we will delineate the general framework of this antiviral response that may have evolved to protect the host against viruses that blunt pathogen-sensing within infected cells.
Additionally, it may have practical importance by identifying a new vulnerability in the life cycle of these human life-threatening viruses that might be targeted for therapeutic purposes. In this respect, we expect that the outcome of the studies of this ANRS JCJC project will result in the discovery of key cellular factors and immune pathways involved in HCV and DENV propagation. This will potentially lead to the identification of novel therapeutic targets that could be used for development of new molecules or therapeutic approaches.
Finally, this project will also contribute to define the regulatory mechanisms of exosomes biogenesis that remains poorly understood.

The works on the aim 2 of the proposal already leads to a publication of an original article (name of lab member is underlined):
Décembre E*, Assil S*, Hillaire MLB, Dejnirattisai W, Mongkolsapay J. Screaton GR, Davidson A and Dreux M. Sensing of Dengue virus infected cells producing immature particles induces an antiviral response by plasmacytoid dendritic cells. PLoS Pathog. 2014 Oct 23;10(10):e1004434.

Our work in this specific area also triggers invitations for review articles/commentaries:
- Assil S, Webster B, Dreux M. Regulation of the host antiviral state by intercellular communications. Viruses. In press.
- Cosset FL, and Dreux M. 2014. HCV transmission by hepatic exosomes establishes a productive infection. J Hepatol 60:674-675
- Assil S, Dreux M. Modulation of permissiveness and antiviral response against hepatitis C virus by interferon lambda-associated polymorphism Med Sci. 2014. 30:1073-5.
- Assil S, Dreux M. Exosomes are carriers for immunostimulatory viral RNA. Med Sci. 2013 29:104-6.

This work led to invitations for oral presentations (M. Dreux) in international and national meetings:
- Journée Jean-Claude Dreyfus de l'Institut Cochin (Paris, Sept. 2015)
- EMBO meeting: human RNA viruses (Istanbul, Oct. 2014)
- Congrès de la Société Française de Microbiologie (Paris, April 2014).

Viral nucleic acids, recognized within the infected cells, trigger an antiviral response characterized by the production of type I interferons (IFNs) and IFN-stimulated genes (ISGs) that suppress viral spread. Many viruses, including hepatitis C virus (HCV) and dengue virus, have thus evolved potent mechanisms that preclude IFN response within infected cells. Nonetheless, these pathogens strongly trigger the expression of ISGs in infected humans, suggesting the existence of alternative pathogen-sensing mechanisms. Consistently, very recently, we demonstrated that HCV infected cells secrete viral RNA-containing exosomes that efficiently transfer immunostimulatory viral RNAs to professional IFN-producing plasmacytoid dendritic cells (pDCs) which, in response, produce robust levels of IFN (Dreux et al. 2012). Importantly, we have already identified key cellular factors required for pDC activation, including the “endosomal sorting complex required for transport” (ESCRT) proteins, required for multivesicular bodie (MVB) formation and exosome biogenesis, and Annexin A2 (ANXA2). Interestingly, ANXA2 is a multifunctional factor involved in various mechanisms such as e.g., the regulation of membrane dynamics. Indeed, ANXA2 is a phospholipid-binding protein that regulates actin assembly. ANXA2 has a dual localization at the plasma membrane and endosomes, where it regulates membrane remodeling and endosome maturation respectively. In Work-Package 1, we will define the regulatory mechanisms of this newly discovered antiviral response triggered by HCV infected cells. Specifically, we will first determine how ANXA2 controls the biogenesis, trafficking and/or release of HCV RNA-enriched exosomes and which of its cellular function(s) are required (Task 1). Second, ANXA2 is known to be recruited to HCV replication complexes via the domain III of the HCV NS5A protein. Our preliminary results suggest that this domain is also required to trigger pDC IFNa production. We will thus determine how ANXA2 and NS5A orchestrate the biogenesis and trafficking of the viral RNA produced at HCV replication complexes – that are included in membrane structures derived from endoplasmic reticulum – to cellular factor(s) and/or compartment(s) permitting its encapsulation and secretion into immunostimulatory exosomes (Task 2). Finally, we will analyze how HCV RNA-exosomes transmission to the pDCs is facilitated by cell-cell contact (Task 3). Specifically we will define the cell surface factor(s) that favor a short-range transfer by concentrating exosomes at the cell surface.
Interestingly, our unpublished results suggest that cells that are infected by dengue virus (DENV) similarly trigger a potent IFN response by pDCs in an exosome-dependent manner. We will thus study the molecular basis of pDC activation by DENV infected cells (WP2), by determining i/ the contribution of exosomes as carrier for cell-to-cell transmission of DENV immunostimulatory RNA and the modalities of pDC response (Task 4) and ii/ the cellular factors required for the biogenesis of the pDC activating signal in DENV infected cells (Task 5).
In conclusion, this ANR JCJC proposal aims at defining, with multidisciplinary approaches, the series of host-virus interaction events that permit the biogenesis within HCV and DENV infected cells of this previously unrecognized carrier of immunostimulatory RNA. By deciphering shared and specific host-virus interplays using evolutionary divergent viruses, we will delineate the general framework of this antiviral response that may have evolved to protect the host against viruses that blunt pathogen-sensing within infected cells. Additionally, it may have practical importance by identifying a new vulnerability in the life cycle of these human life-threatening viruses that might be targeted for therapeutic purposes. Finally, this project will also contribute to define the regulatory mechanisms of exosomes biogenesis that remains poorly understood.