Stem-loop structures II-IV of the 5? untranslated sequences are required for the expression of the full-length hepatitis C virus genome

Abstract

The 5' and 3' untranslated regions (UTR) of the hepatitis C virus (HCV) genome contain stem-loop structures, which are important in viral gene expression and replication. In this study, the functional roles of the predicted stem-loop structures of HCV 5' UTR and 3' UTR in viral gene expression were examined using a chimeric clone of full-length HCV genomic cDNA clone and the gene for green fluorescent protein (GFP). High level expression of the HCV-GFP chimera in Huh-7 cells was accomplished by using a replication defective adenovirus that expresses T7 RNA polymerase and transcription plasmid containing full-length HCV-GFP chimera under the control of a T7 promoter. The HCV-GFP clone, with deletion of stem-loop I, expressed proteins in transfected Huh-7 cells at comparable levels to the wild type HCV clone. Other mutations of the 5' UTR, which either deleted or altered the base pairing of stem-loops II to IV, completely abolished the expression of HCV-GFP chimera. In contrast, deletion of 3' UTR sequences had no effect on HCV protein expression. These findings suggest that the stem-loop structures II to IV of HCV 5' UTR are necessary for protein expression, but that stem loop I is dispensable for protein translation. The stem-loop structures of 3' UTR of HCV genome appear to have no direct role in viral gene expression.

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

“…The genome of HCV is organized into a highly conserved 5’-untranslated region (5’ UTR), a large open reading frame (ORF) and a 3’-untranslated region (3’ UTR). The 5’ UTR of HCV genome binds to the host ribosome using the internal ribosome entry site (IRES) mechanism that facilitates translation of HCV protein [3,4]. The HCV genome contains a large open reading frame (ORF) that encodes for a polyprotein 3011 amino acid long.…”

Interferons α, β, γ each inhibit hepatitis C virus replication at the level of internal ribosome entry site‐mediated translation

“…We thought that inhibition of cap – as well as IRES‐dependent translation inhibition of luciferase by IFN could have occurred because of type of dicistronic expression system used here. As an alternative to this, we used a chimeric HCV cDNA clone where the gene for the green fluorescence protein was inserted in frame with core protein (38, 39). High‐level expression of the HCV core‐GFP fusion protein was achieved in Huh‐7 cells using a low amount of replication defective adenovirus that expresses T7 RNA polymerase.…”

“…Since the former approach does not allow direct visualization of gene expression from the IRES in the transfected cells, we employed an alternative approach that involves stable expression of fluorescence protein from the IRES sequence. We have used a chimeric HCV clone fused to green fluorescent protein (HCV‐GFP) that we had previously constructed in our laboratory (38, 39). High level expression of core‐GFP fusion proteins in Huh‐7 cells was achieved by using a two‐step transfection procedure that involves the use of replication defective adenovirus that expresses T7 RNA polymerase.…”

SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication

“…For example, PCBP regulates translation-replication switch in poliovirus life cycle (Back et al, 2002;Gamarnik and Andino, 1998). It was reported previously that stem-loop I and II are critical for HCV RNA replication, and stem-loop II, III, and IV are important for HCV RNA translation (El-Hage and Luo, 2003;Fukushi et al, 2001;Qi et al, 2003). La autoantigen was shown to bind to loop IV of HCV 5′NTR (Ali and Siddiqui, 1997).…”