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Home > Departments > Virology > Didier PONCET : Molecular Biology of Rotaviruses

Didier PONCET : Group Presentation

-  Rotavirus is the primary cause of severe dehydrating diarrhea in infants and young children. Rotavirus infections result in more than 500,000 deaths worldwide each year (the vast majority occurring in children under 5 in developing countries). Rotavirus infections also cause significant economic losses in agriculture. Despite their pathogenic importance, many steps of the replication cycle of these viruses are not understood.
-  Our aim is to acquire knowledge about the molecular biology of Rotaviruses.

-  Rotavirus, a major cause of gastroenteritis in young mammals, is responsible for hundreds of thousands deaths each year worldwide. Rotavirus infections also cause large economic losses in agriculture. Despite their importance in human and animal health, many steps of the viral life cycle are poorly understood. By the originality of its genome (segmented double stranded RNA) and the characteristics of its cycle, rotavirus is an excellent model to address basic biological questions; entry of virus into the cell, the modification of cellular genes expression (transcription and translation) and selective recognition of molecules (Packaging of eleven dsRNA molecules in each viral capsid), the functionning of molecular machine (transcription ) ...
- The "rotavirus" team lead basic research aimed at understanding rotavirus at different levels.
- 1 / its interaction with the host , most particularly the regulation of translation of viral and cellular mRNA.
-  Rotavirus protein NSP3 interacts with the translation initiation factor eIF4G and simultaneously with the 3 ’end of the rotavirus mRNAs promoting their translation.
- We have developed an RNA electroporation and RT-qPCR assay to quantify the effect of NSP3 mutant on the translation of pseudo-viral RNA. We have shown that whereas rotavirus infection stimulates the expression of pseudo-viral RNA and decreases the expression of poly(A) mRNAs, the expression of NSP3 protein alone did not alter the expression of poly(A) mRNAs, but increases the translation of viral RNA over 100 times. Analysis by RT-qPCR showed no RNA stabilization by NSP3. By using NSP3 mutated either in the RNA- or the eIF4G- binding domain, we showed that the translation increase of viral mRNAs requires complete NSP3. So, contrary to what had been suggested by others, the increase in viral RNA translation by NSP3 is not du to a mere stabilization of viral RNAs and the two functional domains of NSP3 are not independent.

-  - 2 / replication and packaging of the segments via
- A / the development of reverse genetics
- In collaboration with Antoine-Garbarg Chenon (Inserm U538) we developed a technique for the introduction of cloned rotavirus genes in infectious viruses (reverse genetics) without selection pressure (Troupin et al, 2010.). But this selection method causes significant genomic rearrangements (Duponchel et al., 2014). So we try to develop a technique using either a thermo-sensitive assistant rotavirus (following the protocol developed by J. Patton (Trask et al., 2010)) or directly from all the eleven cloned viral genes .
-  - B / the study of non-structural proteins (NSP2, nsP5).
- Rotavirus replication occurs in cytoplasmic inclusions (viroplasms) formed by NSP5 and NSP2 proteins in complex with the viral polymerase VP1 to form a replication complex. The crystallographic structure of NSP2 is known but not that of NSP5. Analysis of recombinant NSP5 showed that its N-terminal half is little structured and that its C terminus allows the formation of decametres and interacts with NSP2 (Martin et al, 2010;. Martin et al, 2011.). We have shown that a dimer of NSP5 binds an iron atom via two highly conserved cysteines (Martin et al., 2013). The role of this iron atom in the functioning of the protein is still a matter of speculation but it can be noted that some polymerases subunits, or enzymes involved in nucleosides metabolism also contain iron atoms.


PONCET Didier [Senior Researcher - INRA]
Molecular Biology of Rotaviruses [Leader]
01 69 82 38 35 Gif - Bât 14

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