title: Translation of Sindbis Subgenomic mRNA is Independent of eIF2, eIF2A and eIF2D.
abstract: Translation of Sindbis virus subgenomic mRNA (sgmRNA) can occur after inactivation of eIF2 by phosphorylation in mammalian cells. Several studies have suggested that eIF2 can be replaced by eIF2A or eIF2D. HAP1 human cell lines knocked-out for eIF2A, eIF2D or both by CRISPR/Cas9 genome engineering were compared with wild-type (WT) cells to test the potential role of eIF2A and eIF2D in translation. Sindbis virus infection was comparable between the four cell lines. Moreover, synthesis of viral proteins during late stage infection was similar in all four cell lines despite the fact that eIF2α became phosphorylated. These findings demonstrate that eIF2A and eIF2D are not required for the translation of sgmRNA when eIF2α is phosphorylated. Moreover, silencing of eIF2A or eIF2D by transfection of the corresponding siRNAs in HAP1 WT, HAP1-eIF2A-and HAP1-eIF2D-cells had little effect on the synthesis of viral proteins late in infection. Modification of AUGito other codons in sgmRNA failed to abrogate translation. Sindbis virus replicons containing these sgmRNA variants could still direct the synthesis of viral proteins. No significant differences were found between the cell lines assayed, suggesting that neither eIF2A nor eIF2D are involved in the translation of this sgmRNA bearing non-AUG codons.
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Universidad Autónoma de Madrid, 28049, Madrid, Spain.
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Sanz
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Universidad Autónoma de Madrid, 28049, Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: González Almela
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Universidad Autónoma de Madrid, 28049, Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Carrasco
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D005057:Eukaryotic Cells / Q000166:cytology / Q000821:virology; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012845:Sindbis Virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C000622391:eIF2D protein, human