Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes.
BACKGROUND
The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance.


RESULTS
Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains.


CONCLUSIONS
These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.
Prabhu
Ramesh
R
Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA. rprabhu@tulane.edu
Garry
Robert F
RF
Dash
Srikanta
S
D020121:5' Untranslated Regions; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004274:DNA, Recombinant; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D016174:Hepacivirus / Q000187:drug effects*; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects*; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000096:biosynthesis
D020121:5' Untranslated Regions; D000998:Antiviral Agents; D004274:DNA, Recombinant; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014758:Viral Core Proteins; C485184:enhanced green fluorescent protein; C069130:nucleocapsid protein, Hepatitis C virus; D049452:Green Fluorescent Proteins