Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs

Abstract

BackgroundHepatitis 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.Materials and methodsThe 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.ResultsThe 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.ConclusionsOverall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

“…The current research demonstrates that the highly conserved 5 ′ UTR of the EV71 genome can be targeted for inhibition of EV71 replication by efficient siRNAs. The results were consistent with previous studies of siRNAs targeting the 5 ′ UTR of other RNA viruses such as hepatitis C virus [ 37 , 38 ] and coxsackievirus B3 [ 20 ]. These studies demonstrated that siRNAs targeting the 5' UTR of the viral genome can exert significant antiviral activity.…”

Hepatitis C Virus Translation Inhibitors Targeting the Internal Ribosomal Entry Site

“…54, 63–72 The design principle shared in common by these constructs requires an accessible single stranded sequence in the IRES 73 that provide the target for a complementary oligonucleotide ligand which is either associated with or, in the case of siRNA and shRNA, directs a ribonuclease activity. Among ribozymes tested in vitro were hammerhead constructs targeted at the apical loop of subdomain IIIb (HCV-195) 52, 53 or the subdomain IIIf (HH363).…”

Silencing HCV Replication in Its Reservoir

“…Viral translation is mediated through IRES site found within the 5 / UTR that consists of ~341 bp in length with highly conserved sequence even between different HCV isolates. IRES can initiate viral polyprotein translation in a cap-independent manner via independently binds to the 40S ribosomal subunit and directs the ribosome to the initiation codon of mRNA of HCV to expedite translation in a cap-independent manner and it is a vital target for the development of new antiviral compounds [ 15 ].…”

“…It contains four profoundly organised stem-looped domains that named I-IV which facilitate HCV RNA translation. Domain I is not essential for IRES activity but rather essential for HCV replication, IRES in domains II-IV play a role in viral genetic replication in a cap-independent manner while Domain III contains subdomains that are critical for the binding of 40S ribosomal subunit [ 6 ]. Translation of HCV mediated by a profoundly conserved internal ribosomal Entry site (IRES) within the 5’UTR making it a significant focus for new medication development [ 5 ].…”