title: The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function.
abstract: Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES-40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES-RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA-protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES-RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S-3' UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES-RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Prasanna
authors.initials: P
authors.lastname: Bhat
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Shivaprasad
authors.initials: S
authors.lastname: Shwetha
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Divya Khandige
authors.initials: DK
authors.lastname: Sharma
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Agnel Praveen
authors.initials: AP
authors.lastname: Joseph
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Narayanaswamy
authors.initials: N
authors.lastname: Srinivasan
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India sdas@mcbl.iisc.ernet.in.
authors.forename: Saumitra
authors.initials: S
authors.lastname: Das
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D012269:Ribosomal Proteins; C030831:ribosomal protein S5