Download Proposition stage ENS 2017

MASTERBIOSCIENCES
ECOLE NORMALE SUPERIEURE DE LYON
Offre de stage de Master / Master Internship offer M2
Host laboratory:
CIRI, INSERM U111, ENS Lyon.
Team VIV (Vesicular trafficking, Innate response & Viruses)
Internship supervisor:
Marlène Dreux (Team leader), Email: [email protected], Phone: 04 26 23 38 34
Website: http://ciri.inserm.fr/en/team/
Research project title:
Analysis of the activation of innate immunity via short-range transfer of viral
genome by non-infectious carrier.
Figure: Working model of
IFN production by pDCs
triggered by sensing of
immature non infectious
particles
produced
by
DENV infected cells.
Project description:
Context: The dengue virus (DENV) is a RNA virus that causes the most prevalent arthropodborne viral disease with an estimated 390 million human infections yearly. Global warming
increases the dissemination of mosquitoes, which are the vectors for DENV, and therefore
augments the risks of infection. DENV infection in humans causes dengue fever and severe
pains, along with life-threatening forms of hemorrhagic fever and shock syndrome. Neither
therapeutic nor vaccine is currently available.
In the lab, we study the activation of the innate immunity against DENV infection. This
response is critical for the control of DENV infection, but also plays a pivotal role in DENV
pathogenesis, by leading to many of the correlates of DENV-associated disease, including
hemorrhaging. Yet, innate immune response is known to be potently inhibited by viral proteins
within infected cells, thus the regulation of such response is still poorly understood.
We recently uncovered that the sensing of cells infected by DENV greatly induces innate
immune response by immune cells that have specialized function for type I interferon (IFN)
production, the plasmacytoid dendritic cells (pDCs). Type I IFNs (i.e., IFNα and β) are key
players leading to an antiviral state, which blocks viral spread. These IFNs also impart the
adaptive immunity. We showed that pDCs are non-permissive to DENV infection. Thereby, this
sensing and production of IFN by pDCs bypasses signaling blockade by viral proteins. We
further demonstrated that pDC activation required the establishment of polarized cell-to-cell
contacts between DENV infected cells and pDCs.
During the production of DENV virions, the newly formed viral particles are known to undergo
maturation in the secretory pathway by a furin-mediated cleavage of the prM surface viral
protein into M cleaved form. This step is required for the fusogenic activity of the surface viral
MASTERBIOSCIENCES
ECOLE NORMALE SUPERIEURE DE LYON
proteins, thus rendering the particles competent to infect cells. Nonetheless, a large number of
immature particles (i.e., non infectious viral particles containing uncleaved prM) is released
from DENV infected cells as a consequence of an evolutionary conserved suboptimal cleavage
site. The release of immature virions is an important feature observed for the different
members of the flavivirus genus, as well as for viruses from various other families.
Importantly, we demonstrated that cells producing immature particles (i.e., the noninfectious virions) are more potent at inducing the pDC activation than cells producing
mature infectious virus. Additionally, we showed that pDC IFN production by DENV infected
cells involves TLR-7, a RNA immune sensor localized in the endo-lysosomal
compartment. The membrane fusion of DENV virions, leading to the release of viral genome
in the cytoplasm, is known to occur at acidic pH, implying that this event also takes place
within in the endo-lysosome compartment. Therefore, we proposed a working model, in which
the immature virions (i.e., incompetent for membrane fusion) are likely retained in the endolysosome compartment, resulting in a potent TLR-7-mediated activation of the IFN production
by the pDCs (Figure). In sharp contrast, we showed that cells producing more mature particles
are poor inducers of pDC IFN production, likely because the fusion-competent mature particles
can escape from this compartment by membrane fusion. Therefore, these particles could
evade from the TLR7 recognition.
We hypothesis that this newly uncovered concept might have a broader importance for the
many viruses that, like DENV, can prevent the pathogen-sensing machinery within infected
cells and that release large number of virus with uncleaved glycoproteins.
Objectives: The proposed project for a Master’s student is, accordingly, to study the
mechanism by which pDC senses DENV infected cells. Especially, we propose to define the
regulation of pDC activation by the membrane fusion ability of the virions, by testing mutants
impaired for this function. In the long term, we will determine the importance of this newly
discovered concept for evolutionary distant viruses. In particular, we will study the activation
of pDCs by viral particles that contain uncleaved surface viral proteins in the context of
viruses that are: i/ known to undergo maturation in the extracellular milieu and ii/ to possess a
pH dependent-cell entry by endocytosis.
Methods: This M2 project will involve the use of molecular biology techniques (i.e., cloning and
mutagenesis), cell biology (i.e., culture of cell lines and/or primary cells, flow cytometry), and
biochemistry (i.e., Western blotting and ELISA).
Selected publications on the topic by the host Team:
1. Webster B, Assil S, Dreux M. Cell-cell sensing of viral infection by pDCs. 2016. J Virol in
press
2. Assil S, Webster B, Dreux M. Regulation of the Host Antiviral State by Intercellular
Communications. 2015. Viruses. 7:4707-33.
3. Décembre, E., S. Assil, M. L. B. Hillaire, W. Dejnirattisai, J. Mongkolsapaya, G.R. Screaton,
A.D. Davidson, and M. Dreux. 2014. Sensing of immature particles produced by dengue virus
infected cells induces an antiviral response by plasmacytoid dendritic cells. PLoS Pathogens
10:e1004434
4. Cosset, F. L., and M. Dreux. 2014. HCV transmission by hepatic exosomes establishes a
productive infection. J Hepatol 60:674-675.
5. Dreux*, M., U. Garaigorta, B. Boyd, E. Decembre, J. Chung, C. Whitten-Bauer, S. Wieland,
and F. V. Chisari*. 2012. Short-range exosomal transfer of viral RNA from infected cells to
plasmacytoid dendritic cells triggers innate immunity. Cell Host Microbe 12:558-570.