Unit: Enterotropic viruses and antiviral strategies
Director: Colbère-Garapin Florence
The field of study of the laboratory concerns human enterotropic viruses (enteroviruses and rotavirus) and antiviral strategies, in particular RNA interference. Our project focuses on various aspects of the cellular response to viral infection, such as the resistance of cells to persistent viral infection and apoptosis. We are also studying emerging, circulating viruses, involved in recent poliomyelitis epidemics (PTR 120, leader : Francis Delpeyroux, collaboration between IP Paris and IP Madagascar).
Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells. Labadie, K., Pelletier, I., Saulnier, A., Martin, J. & Colbère-Garapin, F. Virology, 2004, 318, 66-78.
Immunodeficient patients whose gut is chronically infected by poliovirus (PV) may excrete large amounts of virulent virus for years. To investigate how PV establishes chronic infections in the gut, we tested whether it is possible to establish persistent PV infections in human intestinal cells. Four PV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost two years (J. Martin et al., J. Virol., 2000, 74, 3001), were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A PV mutant of the first isolate excreted by the patient was, like the vaccinal Sabin 3 strain, lytic in both types of intestinal cell cultures. In contrast, the three other PV mutants tested, excreted between a few and 18 months later, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected in the patient conferred on PV mutants the capacity to infect intestinal cells persistently. The establishment of persistent infections in enterocytes was not due to poor replication of PV in these cells, but was associated with reduced viral adsorption to the cellular receptor CD155. The percentage of cultures persistently infected by PV was higher for enterocytes than for undifferentiated cells. This implicates cellular determinants of cell cycle arrest and/or differentiation of enterocytes in persistent PV infections.
Cure of persistently PV-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 in vitro and in vivo models of acute infection: small interfering RNAs (siRNAs) induce sequence-specific degradation of targeted mRNA. 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. 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 the laboratory. Cultures of human cells infected for several months by a persistent PV mutant were treated with specific antiviral 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 apoptosis. A. Wirotius, A. Autret, S. Martin-Latil, 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 the virus can persist in the central nervous system (CNS) of paralyzed animals. PV persistence could be due, at least in part, to an inhibition of viral genome synthesis in the CNS (Girard S. et al., J. Gen. Virol., 2002, 83, 1087). Otherwise, during the acute phase of poliomyelitis we observed that motor neurons died by an apoptotic process.
To investigate PV-induced apoptosis in nerve cells, we have developed mixed mouse primary nerve cell cultures from the cerebral cortex of neonatal mice (Couderc T. et al., J. Gen. Virol., 2002, 83 1925). We showed that PV-induced apoptosis involved both caspase activation and mitochondrial dysfunction associated notably with a drop in the mitochondrial transmembrane potential and the translocation from mitochondria to the cytosol of apoptotic factors including cytochrome c. 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 could play a role in PV persistence in neural cells.
We are currently studying signaling pathways involved in this process in a human neuronal cell model. In healthy cells, the pro-apoptotic protein Bim (Bcl-2 interacting mediator of cell death), by interacting with the dynein complex, is usually maintained in an inactivated form associated with the cytoskeleton. We showed that Bim is released to the cytosol following PV infection. This result is particularly interesting since the cytoplasmic domain of CD155 also interacts with the dynein complex. Possibly, Bim is released following PV-CD155 interaction such that it could neutralize Bcl-2, thereby leading to apoptosis. This hypothesis is currently being investigated.
Keywords: enterovirus, rotavirus, siRNA, antiviral, apoptosis, persistent infection