title: Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs.
abstract: BACKGROUND
  Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.
  
  
  METHODS
  The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.
  
  
  RESULTS
  The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.
  
  
  CONCLUSIONS
  Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.
authors.affiliation: Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. sabahat711@yahoo.com
authors.forename: Saba
authors.initials: S
authors.lastname: Khaliq
authors.forename: Shah
authors.initials: S
authors.lastname: Jahan
authors.forename: Asim
authors.initials: A
authors.lastname: Pervaiz
authors.forename: Usman
authors.initials: U
authors.lastname: Ali Ashfaq
authors.forename: Sajida
authors.initials: S
authors.lastname: Hassan
mesh_terms: D020121:5' Untranslated Regions; D000998:Antiviral Agents / Q000494:pharmacology; D001688:Biological Products / Q000494:pharmacology; D002460:Cell Line; D020868:Gene Silencing; D017930:Genes, Reporter; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D010154:Pakistan; D014176:Protein Biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D019562:Viral Load; D014779:Virus Replication / Q000187:drug effects
chemical_list: D020121:5' Untranslated Regions; D000998:Antiviral Agents; D001688:Biological Products; D034741:RNA, Small Interfering; D012367:RNA, Viral; D049452:Green Fluorescent Proteins