Unit: Enterotropic viruses and antiviral strategies

Director: Colbère-Garapin Florence

The laboratory works on human enterotropic viruses (enteroviruses and rotavirus) and antiviral strategies. Our project focuses on various aspects of the cellular response to viral infection, including signalling pathways leading to either cell survival or cell death. We are also studying emerging, circulating, recombinant enteroviruses, and particularly those involved in recent poliomyelitis epidemics in Madagascar (PTR 120, leader: Francis Delpeyroux, collaboration between IP Paris and IP Madagascar).

Mechanisms of resistance to virus-induced cytopathy. K., Labadie, A., Saulnier & F.Colbère-Garapin.

We have previously shown that viral determinants selected in a hypogammaglobulinaemic patient conferred on poliovirus (PV) mutants the capacity to infect human intestinal cells persistently. The establishment of persistent infections in intestinal cells is associated with reduced viral adsorption onto the cellular receptor CD155. Cellular determinants of cell cycle arrest and/or differentiation of enterocytes were also implicated in the mechanism of these persistent PV infections (Labadie, K., Pelletier, I., Saulnier, A., Martin, J. & Colbère-Garapin, F. Virology, 2004, 318, 66-78). Some cell cultures spontaneously stopped producing virus after several months of persistent PV infection and no viral RNA was detected in the cured cells. Two human intestinal cell cultures cured of persistent PV infection were susceptible to de novo PV infection, but partially resistant to PV-induced apoptosis, whereas parental cells were susceptible to both infection and apoptosis. This simple model is therefore useful for elucidating the molecular mechanisms of resistance to virus-induced cytopathy.

Cure of persistently virus-infected cells by specific siRNAs. A. Saulnier, I. Pelletier, K. Labadie & F . Colbère-Garapin.

Post-transcriptional RNA interference is now widely used as an antiviral strategy in both in vitro and in vivo models of acute infection: small interfering RNAs (siRNAs) induce sequence-specific degradation of the mRNA targeted. SiRNAs targeting various regions of the PV genome reduce the viral yield by a factor of 10 to 100 after infection of human cells by a lytic PV strain (chemical synthesis of siRNAs: C. Gouyette, PT7; or Proligos). Inhibition of viral multiplication is specific: it does not occur without siRNA or with a non-specific, control siRNA. We tested the effect of siRNAs in a model of persistent PV infection developed in our laboratory. Cultures of human cells infected for several months by a persistent PV mutant were treated with specific anti-PV siRNAs. The majority of persistently infected cultures stopped producing virus after this specific treatment, whereas the cure of cultures by non-specific siRNAs was a rare event. The complete cure of cells by antiviral RNAs was confirmed by the most sensitive techniques available. These results demonstrate that antiviral siRNAs can cure cell populations persistently infected for several months.

CD155 and poliovirus-induced neuronal apoptosis. A. Autret, S. Martin-Latil, A. Wirotius, L. Mousson, I. Pelletier, F. Colbère Garapin & B. Blondel. Collaboration with J. Estaquier, F. Petit & D. Arnoult.

We have previously shown, using a mouse model experimentally infected with PV, that infected motor neurons die by an apoptotic process and that the virus can persist in the central nervous system (CNS) of paralyzed animals. Our results also indicate that the interaction between PV and its receptor, CD155, may modulate apoptosis according to the allelic form of CD155 expressed at the cell surface (Gosselin A. S., Simonin Y. et al., Journal of Virology, 2003, 76, 790). This modulation may play a role in PV persistence in neuronal cells.

We are currently studying pro-apoptotic signalling pathways induced by PV in human neuronal cells, neuroblastoma IMR-5 cells. Our results suggest that the mitochondrial apoptotic pathway is involved in PV-induced apoptosis in IMR-5 cells, through a Bax-dependent mechanism. They also suggest that the pro-apoptotic " BH3-only " protein Bim (Bcl-2-interacting mediator of cell death), usually maintained in an inactivated form associated with the cytoskeleton by interacting with the dynein complex, is released into the cytosol following PV infection. This result is particularly interesting because the cytoplasmic domain of CD155 also interacts with the dynein complex. Possibly, Bim is released following PV-CD155 interaction such that it could either neutralize the anti-apoptotic protein Bcl-2, or activate Bax, thereby leading to apoptosis.

Role of cell signalling in rotavirus pathogenicity. S. Martin-Latil, I. Pelletier, L. Mousson, B. Blondel & F. Colbère-Garapin.

Rotaviruses are responsible for one third of the cases of severe gastroenteritis in young children worldwide, and cause more than 500 000 deaths a year among these children in developing countries. We are studying the activation of cell signalling pathways following interactions between rotaviruses and integrins, their cellular receptors. In particular, we are focusing on pathways leading to epithelial cell death and contributing to rotavirus-induced pathology.

Keywords: enterovirus, rotavirus, siRNA, antiviral, apoptosis, persistent infection, virology


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