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Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model.

ABSTRACT: Ribavirin (RBV) is a broad-spectrum antiviral active against a wide range of RNA viruses. Despite having been used for decades in the treatment of chronic hepatitis C virus (HCV) infection, the precise mechanism of action of RBV is unknown. In other viruses, it inhibits propagation by increasing the rate of G-to-A and C-to-U transitions. Here, we utilized the J6/JFH1 HCV cell-culture system to investigate whether RBV inhibits HCV through the same mechanism. Infected Huh7.5 cells were treated with increasing concentrations of RBV or its phosphorylated forms. A fragment of the HCV NS5B-polymerase gene was amplified, cloned, and sequenced to estimate genetic distances. We confirm that the antiviral effect of all three RBV-drug forms on HCV relies on induction of specific transitions (G-to-A and C-to-U). These mutations lead to generation of non-infectious virions, reflected by decreased spread of HCV in cell culture despite relatively limited effect on virus genome titers. Moreover, treatment experiments conducted on a novel Huh7.5 cell line stably overexpressing adenosine kinase, a key enzyme for RBV activation, yielded comparable results. This study indicates that RBV action on HCV in hepatoma cell-culture is exerted through increase in mutagenesis, mediated by RBV triphosphate, and leading to production of non-infectious viruses.

SUBMITTER: Galli A

PROVIDER: S-EPMC5854589 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model.

Galli Andrea A Mens Helene H Gottwein Judith M JM Gerstoft Jan J Bukh Jens J

Scientific reports 20180315 1

Ribavirin (RBV) is a broad-spectrum antiviral active against a wide range of RNA viruses. Despite having been used for decades in the treatment of chronic hepatitis C virus (HCV) infection, the precise mechanism of action of RBV is unknown. In other viruses, it inhibits propagation by increasing the rate of G-to-A and C-to-U transitions. Here, we utilized the J6/JFH1 HCV cell-culture system to investigate whether RBV inhibits HCV through the same mechanism. Infected Huh7.5 cells were treated wit ...[more]

PMID: 29545599

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Project description:Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy. In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4'-diisothiocyano stilbene-2,2'-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

Project description:UnlabelledThe first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively.ImportanceHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

Dataset Information

Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model.

Publications

Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model.

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