1 Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA
2 Department of Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA
3 Department of Microbiology and Immunology, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA
4 Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK
Correspondence
Srikanta Dash
sdash{at}tulane.edu
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ABSTRACT |
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A table of expression data, and two figures showing a ribonuclease protection assay and flow analysis of GFP expression are available as supplementary material in JGV Online.
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MAIN TEXT |
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The genomic organization of all HCV strains is similar. All have a 5' untranslated region (UTR), a large open reading frame (ORF) and a 3'UTR. The sequences present in the 5'UTR region form a highly ordered structure that can attach to host cell ribosomes and initiate translation by an internal ribosome entry site (IRES)-mediated mechanism. This type of cap-independent mechanism is known to be operative in certain other RNA viruses including picornaviruses (Collier et al., 1998; Saiz et al., 1999
; Castet et al., 2002
), and differs in several aspects from cap-dependent translation (Kozak, 2003
). According to the reports of several different laboratories nt 40370 of the HCV RNA genome are important for the IRES-mediated translation (Tang et al., 1999
; Ali et al., 2000
; Dasgupta et al., 2004
). Therefore, sequence variability of the 5'UTR has important implications for the structural organization and function of the IRES element (Saiz et al., 1999
). The RNA genome binds to the host cell ribosome and is translated to yield a large polyprotein of 3010 aa. This polyprotein is then cleaved intracellularly by the combined action of cellular and virally encoded proteases. Ten different mature proteins are generated. The structural proteins, core, E1 and E2 are required for virus assembly, export and binding to cellular receptors for infection. The non-structural proteins encode enzymes required for replication of the viral genome. Following the stop codon, the viral genome ends with sequences of variable length among different HCV genotypes, with a highly conserved 98 nt segment at the very terminus of the 3'UTR. These sequences are required for the initiation of antigenomic-strand synthesis (Reed & Rice, 2000
). We have reported previously that IFN-
, IFN-
and IFN-
each target the highly conserved 5'UTR of the HCV genome, utilized by the virus to translate protein by an IRES-dependent mechanism (Dash et al., 2005
).
We conducted this study to determine if quantitative differences in the IFN action of inhibiting IRES-mediated translation among different HCV genotypes could explain the viruses differential responses to treatment. We used standard polymerase chain reaction (PCR) and cloning methods to construct chimeric clones between the sequence encoding the green fluorescence protein (GFP) and HCV IRES sequences of different genotypes. High-level expression of GFP from different IRES clones was accomplished in Huh-7 cells using a two-step-transfection procedure. This method allows us to examine cells expressing GFP directly under a fluorescent microscope, without the requirement for immunological detection procedures. To examine the effect of IFN treatment on cap-dependent translation, Huh-7 cells were co-transfected with 1 µg IRES-GFP plasmid DNA along with 1 µg pDsRed2 red fluorescence protein (RFP) plasmid (BD Biosciences Clontech) using the FuGENE 6 (Roche Molecular Biology) transfection reagent. The effect of IFN on translation was determined at 24 h by examining the expression of GFP or RFP under a fluorescence microscope (Olympus), at 484 nm for the expression of GFP, 563 nm for the expression of RFP and 340 nm for DAPI. The percentage of GFP-positive Huh-7 cells was quantitatively measured using CELL QUEST computer software. As a control, we examined the specificity of IFN action by investigating IRES-mediated GFP translation in the presence of other cytokines [interleukin 1 (IL1), IL6, tumour necrosis factor alpha (TNF-) and transforming growth factor beta 1(TGF-
1)]. The stability of IRES-GFP mRNAs in the transfected-cell cultures was compared in the presence and absence of IFN treatment. Total RNA was isolated from IFN-treated cells and the levels of IRES-GFP mRNA were measured by a ribonuclease protection assay (RPA) using a probe specific for the GFP gene.
The IRES clones used in this study include nt 18356 of HCV 5'UTR (Collier et al., 1998). Each clone contained the stemloop IIIV domains, because these sequences are necessary for efficient IRES-mediated translation. These sequences were selected based on reports, which suggested that the first stemloop structure (118) is not necessary for translation, and that the core coding sequences can modulate the IRES-activity of HCV (Tsukiyama-Kohara et al., 1992
; Reynolds et al., 1996
; Wang et al., 2000
). The IRES nucleotide sequences from HCV of six different HCV genotypes (16) and two subtypes (1a, 1b, 2a and 2b) are shown in Fig. 1
(a). The IRES sequences of HCV fold into stemloop structures (IIIV) (Fig. 1b
). As we previously demonstrated (Qi et al., 2003
), any alteration in the primary or secondary structures in the stemloop regions of the IRES severely affects translation efficiencies. Stemloop II encompasses nt 43120, stemloop III encompasses nt 134300 and stemloop IV encompasses nt 300354. Stemloop structures IIA and IIB have 7 nt that display differential variations, 28 nt display variations in stemloop structure IIIAE and 5 nt display variations in stemloop structure IV (Fig 1b
). The sequences of HCV 1a and HCV 1b are highly conserved in this region as compared to other genotypes. Thus, the majority of sequence differences in the IRES region among different HCV genotypes are in stemloop III. We examined whether the nucleotide sequence differences in the stemloop IIIV domains of the HCV IRES affect the expression of GFP in Huh-7 cells or not. We achieved high-level expression of GFP from different IRES clones in Huh-7 cells using a replication-defective adenovirus that expresses T7 RNA polymerase. These high levels of GFP expression from different IRES constructs were comparable, indicating that all the IRES sequences are equally efficient in mediating the translation of GFP, even though they have minor nucleotide sequence variations in the stemloop IIIV regions.
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IFN- induces the transcriptional activation of the 2',5' (A) synthetase, which polymerizes ATP into 2',5'-linked oligoadenylates of variable lengths. This enzyme can activate a latent ribonuclease, Rnase L, present in all animal cells. The activated RNase L can then cleave single-stranded RNAs. We examined the stability of IRES-GFP messages after IFN treatment using an RPA, and found that the levels of IRES-GFP mRNA did not significantly differ in cells with 11000 IU IFN ml1 or without IFN treatment (see Supplementary Fig. S2 in JGV Online). This excludes the possibility that inhibition of GFP expression from the IRES clones is due to extensive degradation of IRES-GFP mRNA. However, our study does not exclude the involvement of RNase L pathways induced by IFN and their role in IFN therapy. We also cannot rule out the possibility that some of the IRES mRNA could have undergone partial hydrolysis by RNase L pathways. A study performed by David Barton's group (Han & Barton, 2002
) indicated that certain HCV genotypes are more sensitive to RNase degradation than others. In their analysis, HCV genotypes 2a, 2b, 3a and 3b were more sensitive to RNase-mediated degradation than HCV 1a and 1b. The RNA of genotypes 2a, 2b, 3a, 3b was degraded to fragments of 2001000 bases in length. We did not observe this preferential degradation in our investigation using IRES clones. Taken together, results from this study suggest that IFN treatment inhibits translation of all IRES mRNA without RNA degradation. Studies are underway to investigate alternative pathways that block translation without degradation of IRES mRNA in IFN-treated cells. We used Huh-7 cells as a model cell line to understand the mechanisms of IFN action against HCV. This cell line efficiently supports HCV replication, and numerous investigators have used this cell line to study the antiviral effects of IFN. It is important that future studies should be performed to confirm these observations using other human hepatic cell lines, including primary human hepatocytes.
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ACKNOWLEDGEMENTS |
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Received 22 April 2005;
accepted 19 August 2005.
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