title: A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.
abstract: We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007.
authors.forename: Swarupa
authors.initials: S
authors.lastname: Panda
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007.
authors.forename: Dhiviya
authors.initials: D
authors.lastname: Vedagiri
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007.
authors.forename: Thangaraj Soundara
authors.initials: TS
authors.lastname: Viveka
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007.
authors.forename: Krishnan Harinivas
authors.initials: KH
authors.lastname: Harshan
mesh_terms: D045744:Cell Line, Tumor; D039561:Eukaryotic Initiation Factor-4E / Q000096:biosynthesis*; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism; D016174:Hepacivirus / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D021381:Protein Transport; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase