Frank Bastian

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

Abstract: Interferon (IFN)‐α is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN‐α, ‐β and ‐γ inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5′ untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full‐length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES‐dependent mechanisms (pCITE‐GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN‐α, ‐β and ‐γ inhibit replication of sub‐genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV‐ and EMCV‐IRES sequences present in the dicistronic HCV sub‐genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES‐mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.

Spiroplasma as a candidate agent for the transmissible spongiform encephalopathies.

The recovery of a novel Spiroplasma sp. from brain tissues from sheep with scrapie, cervids with chronic wasting disease, and from patients with Creutzfeldt-Jakob disease through passage through embryonated eggs has raised the issue of the role of Spiroplasma in the transmissible spongiform encephalopathies (TSE). In this review, we have inserted into an epidemiologic infection model evidence accumulated over the past 30 years showing involvement of Spiroplasma infection in TSE. These data support our hypothesis that a Spiroplasma sp. is the causal agent of TSE, although Kochs postulates must be fulfilled to definitively answer that question.

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

Summary. 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.

Activation of interferon-stimulated response element in huh-7 cells replicating hepatitis C virus subgenomic RNA.

Interferon-alpha (IFNα) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFNα was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFNα. These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.