Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.
Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.
Piron
M
M
Laboratoire de Virologie et Immunologie Moléculaires INRA, CRJJ, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
Vende
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P
Cohen
J
J
Poncet
D
D
D000595:Amino Acid Sequence; D039562:Eukaryotic Initiation Factor-4F; D039603:Eukaryotic Initiation Factor-4G; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010446:Peptide Fragments / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012401:Rotavirus / Q000254:growth & development*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
C113920:EIF4G1 protein, human; D039562:Eukaryotic Initiation Factor-4F; D039603:Eukaryotic Initiation Factor-4G; C082740:NSP3 protein, Rotavirus; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039102:Poly(A)-Binding Proteins; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins