Eukaryotic and Viral Translational Control - CNRS URA 1966  

  HEADDr KEAN Katherine /

  Annual Report

We have been interested in the mechanisms of initiation of protein synthesis in the eukaryotic cell, and on the regulation that can be brought to bear on this. Our work has been particularly focused on the recruitment of the 40S ribosomal subunit to the mRNA, and different viral pathogens have been studied. Our UP closed on December 31st 2006.

Picornaviral Protein Synthesis

Protein synthesis is from an IRES (for Internal Ribosome Entry Site) and for Poliovirus (PV) domain V of this structure is important for virulence, as it contains an attenuating mutation in all the Sabin vaccine strains. We wanted to see whether results concerning PV could be extrapolated to another virus of the same genus but with a different tropism. To this end we used coxsackievirus B3 (CVB3), the most common causal agent of viral myocarditis. As for PV, the Sabin3-like mutation in the CBV3 context results in a significantly reduced viral titer and poor translation efficiency in vitro in rabbit reticulocyte lysates. In contrast to PV, biochemical probing of Sabin-like CBV3 mutant RNA revealed no distinctions from the wild-type.

Hepatitis C virus (HCV) protein synthesis

Again, translation initiation is from an IRES but this time the 3’ end of the RNA is highly structured (the X region). IRES-driven translation usually occurs with the help of specific IRES associated factors (ITAFs), and a pseudocircularisation of the mRNA is likely to be conserved functionally with classical mRNAs, but not mechanistically. We have examined the role of polypyrimidine Tract Binding Protein (PTB) in HCV translation, since its ability to bind both the HCV IRES and the 3’X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure.

Pseudocircularisation model of HCV mRNA dependant on RNA-RNA or RNA-protein interactions for efficient translation initiation.

Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB in 3’X region stimulation of HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrated the involvement of at least two stimulating factors.

Rabies virus (RV) matrix protein as a regulator

Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is multifunctional acting in virus assembly/budding but also in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. We searched for potential RV M protein cellular partners. A yeast two-hybrid screen revealed the eIF3h subunit and the interaction was validated by co-immunoprecipitation and surface plasmon resonance. On expression in mammalian cell cultures, RV M protein was localized in early 40S ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5’-UTRs. Interestingly, translation of HCV IRES-containing mRNA, which recruits eIF3 via a different mechanism, was unaffected. Together, our data suggest that as a complement to its other functions RV M protein inhibits translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.



Publications 2006 of the unit on Pasteur's references database

Activity Reports 2006 - Institut Pasteur
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