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Consensus small interfering RNA targeted to stem-loops II and III of IRES structure of 5? UTR effectively inhibits virus replication and translation of HCV sub-genotype 4a isolates from Saudi Arabia

ABSTRACT: Being the most conserved region of all hepatitis C virus (HCV) genotypes and sub-genotypes, the 5? untranslated region (5? UTR) of HCV genome signifies it’s importance as a potential target for anti-mRNA based treatment strategies like RNA interference. The advent and approval of first small interference RNA (siRNA) -based treatment of hereditary transthyretin-mediated amyloidosis for clinical use has raised the hopes to test this approach against highly susceptible viruses like HCV. We investigated the antiviral potential of consensus siRNAs targeted to stem-loops (SLs) II and III nucleotide motifs of internal ribosome entry site (IRES) structure within 5? UTR of HCV sub-genotype 4a isolates from the Saudi population. siRNA inhibitory effects on viral replication and translation of full-length HCV genome were determined in a competent, persistent, and reproducible Huh-7 cell culture system maintained for one month. Maximal inhibition of RNA transcript levels of HCV-IRES clones and silencing of viral replication and translation of full-length virus genome was demonstrated by siRNAs targeted to SL-III nucleotide motifs of IRES in Huh-7 cells. siRNA Usi-169 decreased 5? UTR RNA transcript levels of HCV-IRES clones up to 75% (P < 0.001) at 24 h post-transfection and 80% (P < 0.001) at 48 h treatment in Huh-7 cells. 5? UTR-tagged GFP protein expression was significantly decreased from 70 to 80% in Huh-7 cells co-transfected with constructed vectors (i.e. pCR3.1/GFP/5? UTR) and siRNA Usi-169 at 24 h and 48 h time-span. Viral replication was inhibited by more than 90% (P < 0.001) and HCV core (C) and hypervariable envelope glycoproteins (E1 and E2) expression was also significantly degraded by intracytoplasmic siRNA Usi-169 activity in persistent Huh-7 cell culture system. The findings unveil that siRNAs targeted to 5? UTR-IRES of HCV sub-genotype 4a Saudi isolates show potent silencing of HCV replication and blocking of viral translation in a persistent in-vitro Huh-7 tissue culture system. Furthermore, we also elucidated that siRNA silencing of viral mRNA not only inhibits viral replication but also blocks viral translation. The results suggest that siRNA potent antiviral activity should be considered as an effective anti-mRNA based treatment strategies for further in-vivo investigations against less studied and harder-to-treat HCV sub-genotype 4a isolates in Saudi Arabia.

SUBMITTER: AlMalki W

PROVIDER: S-EPMC7785429 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Project description:The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

Project description:Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-? ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.

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Consensus small interfering RNA targeted to stem-loops II and III of IRES structure of 5? UTR effectively inhibits virus replication and translation of HCV sub-genotype 4a isolates from Saudi Arabia

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