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  Director : Monique Lafon (mlafon@pasteur.fr)


  abstract

 

Our group is studying the interactions of neurotropic viruses with the nervous system to identify molecular basis of neuron survival. An in vivo approach using several model of transgenic or KO mice, consists in studying the mechanisms developed by pathogenic strains of rabies virus to subvert the host surveillance (strengthen of the immune privilege of the nervous system). An in vitro approach consists in listing and subsequently characterizing the neuron genes utilized by pathogenic virus to subvert the T cell response and to promote intrinsic survival. This is performed by microarrays using human post mitotic neurons infected with herpes Simplex virus and rabies virus of different pathogenicty and by analysing virus gene that control apoptosis.



  report

cale

Upregulation of FasL in the CNS: a mechanism of immune system evasion by rabies virus (Leïla Baloul , Anne Galelli, Serge Camelo, Monique Lafon)

After an injection of mice in the hindlimbs, the highly pathogenic strain of rabies virus CVS invades both spinal cord and brain and causes the death of mice. In contrast, the CNS invasion by the strain of attenuated pathogenicity PV is restricted to the spinal cord and mice survive with paralytic sequels. We found previously that T cells cannot control the neuroinvasive CVS rabies infection. Our data indicate that despite a similar expression of virus and TNF-a mRNAs, the migration of lymphocytes is transitory in fatal rabies, whereas it is sustained in non-fatal rabies. Transitory migration of T cells in fatal rabies, is associated with an increase of cell apoptosis. We found that only fatal rabies upregulates the early expression of FasL mRNAs. FasL is expressed by infected neurons. In mice lacking FasL, (gld) the infection by the neuroinvasive rabies virus strain is less severe, and the number of CD3 T cells undergoing apoptosis is reduced compared to those observed in their normal counterparts. These data strongly support that fatal rabies virus triggers early FasL expression that leads to the destruction of migratory T cells by the Fas/FasL apoptosis pathway. Thus it seems that rabies virus uses an immunosubervise strategy to successfully invade the CNS which takes advantage of the immune privilege status of the CNS.

This could explain why without post-exposure vaccination a rabies virus infection is fatal in most cases.

Rabies virus triggers both caspase-dependent and independent apoptosis in Jurkat T cells (Maria-Isabel Thoulouze, Mireille Lafage, Monique Lafon)

Strains of live rabies virus (RV) vaccine strains triggers of caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect). This process involved caspases, including caspases 3, 8 and 9. Treatment with the pan-caspase inhibitor ZVAD-fmk reduced, but did not abolish, apoptosis, suggesting that a caspase-independent pathway is also induced by RV infection. Indeed, translocation of the apoptosis-inducing factor (AIF) was detected in RV-infected Jurkat T cells. In contrast, strain of RV that does not induce apoptosis did not activate caspases nor translocate AIF. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both caspase activation and AIF translocation, suggesting that Bcl-2 overproduction blocks apoptosis by interacting upstream in both the caspase-dependent and the caspase-independent apoptotic pathway. RV infection and production were similar in Jurkat-vect and in Jurkat-Bcl-2 cells. This indicates that 1) caspase activation is not a prerequisite for RV maturation or budding, and 2), in contrast to what was observed with HIV or Influenza virus infection, Bcl-2 has no direct antiviral effects against rabies virus. Bcl-2 production is naturally upregulated by day 3 in RV-infected Jurkat-vect cultures. This seems to be controlled by the virus infection itself. The Bcl-2 increase results in the establishment of long-term, persistently infected cultures that continue to produce virus particles. Thus, during the course of infection by live RV vaccine strain, Bcl-2-rescued infected cells may therefore be productive reservoirs of virus in the long term.

Pro-apoptotic property of glycoprotein of non-pathogenic rabies virus strain (ERA) (Stéphanie Lay, Christophe Préhaud, Monique Lafon, collaboration with Bernhard Dietzschold, Jefferson Institute, Philadelphia, PA, USA)

We showed that the attenuated live rabies virus (RV) vaccine strain ERA triggers apoptosis in the human neuroblastoma SK-NSH cell line along with activation of caspase, nuclear fragmentation and phosphatidylserine exposure (Photo I). In contrast, infection, with the pathogenic and neurotropic CVS RV strain did not cause apoptosis in Jurkat T cells. Patterns of viral proteins in the cytoplasma and at the membranes are different in CVS and in ERA infection (photo II).To identify which of the two major RV proteins (G and N) is responsible for the triggering of apoptosis, both G and N of ERA were expressed individually in Jurkat T cells by using the inducible Tet-ON expression system. Induction of RV G expression but not RV N expression resulted in apoptosis indicating that the capacity of a particular RV to trigger apoptosis is largely determined by determinants of G protein. To further examine qualitative aspects of RV G that are associated with the induction of apoptosis and to determine whether observations made in Jurkat T cells take place in neurons, we infected human neuronal cells with recombinant RVs in which G from an attenuated strain was replaced by the G from a virulent strain. This experiment revealed that only recombinant RVs containing the G of a non-pathogenic RV strain, but not of a pathogenic strain, are able to trigger apoptosis in neuronal cells. It shows that neither the transcription, nor the replication of the virus can induce major signaling pathways driving the cells to programmed cell death. This suggests that apoptosis is induced by determinants which are only present in the G proteins of non-pathogenic attenuated RV strains. These determinants could be, at least in part, responsible for the unique ability of attenuated RV strains to induce protective immunity.

Apoptotic bodies as powerful immunogens (Christophe Préhaud, Françoise Mégret, Monique Lafon in collaboration with Christophe Batejat and Nicolas Escriou from Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, Paris, France

Intracellular pathogens, such as viruses or intracellular bacteria, often induce apoptosis of the cells they infect. The demise of the infected cells results in the formation of apoptotic bodies which contain cytoplasm along with microbial antigens. Several lines of evidence indicate that these structures are powerful immunogens that stimulate T lymphocytes very efficiently, after their uptake and degradation by antigen presenting cells (APCs: dendritic cells and macrophages) and presentation of microbial antigens via MHC class I or class II molecules.

It now seems clear that the design of new vaccine approaches will gain if we are able to provide professional APCs with sufficient antigen to raise a strong immune response. Indeed, induction of apoptosis has already been exploited as a novel immuno-stimulatory strategy by the introduction of a mutant caspase (the main effector protein involved in apoptosis) in DNA vaccines. Institut Pasteur is one of the largest health research institution in France, with a traditionnal focus on infectious diseases. One of its duty is to generate basic science which can drive to many results which are also suitable for commercialization in the interest of the whole scientific community and for the public good at large. In the laboratory we previously showed that the non pathogenic rabies virus strain ERA was a strong immunomodulator of the B cell response. We established that this immunostimulation was due to the production of apoptotic bodies from ERA virus infected lymphocytes. Subsequently, we proved that the rabies virus glycoprotein of the ERA strain was the key effector of apoptosis induction. These data were patented by Institut Pasteur. Our project was then to take advantage of pro-apoptotic and pro-inflammatory properties of this viral protein to show that its addition to a vaccine formula will increase the B cell response against an heterologous antigen. To answer this question, we decided to improve the imunogenicity of a recombinant pox live vaccine encoding the main antigenic surface protein of Influenza virus.

Micro-arrays of post-mitotic human neurons infected with HSV-1 and rabies virus (Christophe Prehaud, Françoise Megret, Monique Lafon)

We are performing an extensive study the gene expression pattern of neurons upon virus infection. In collaboration with University Louis Pasteur in Strasbourg Genopole we are screening neuron transcriptome by microarrays to identify the gene expression pattern of the infected neuron. Messenger RNA are prepared from synchronized cultures of human post mitotic neuronseither non infected (baseline of non activation) or infected with Herpes simplex virus type 1, HSV-1, or rabies viruses of different pathogenicity.

Photo I : Membranes of rabies virus infected neuroblastoma cell line NK-N-SH expressed rabies virus glycoprotein (A, red) and phosphatidylserine (B, green).

Photo II: Distinct pattern of virus proteins distribution are observed in non-apoptotic virus strain (1) and in pro-apoptotic virus strain (2). Green= rabies virus antigen. Hoechst staining (blue) indicate nucleus fragmentation.

Keywords: Neurology, virology, immunology



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Baran Corinne, part-time secretary (cbaran@pasteur.fr) Lafon Monique, head of unit,mlafon@pasteur.fr

Prehaud Christophe, senior researcher,cprehaud@pasteur.fr

Baloul Leïla, post-doctorant

Lay Stéphanie, PhD student

Bourahoui Amina student

Megret Françoise, Enginer

Lafage Mireille, Technician


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