Ramesh Prabhu

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.

Inhibition of Hepatitis C Virus Nonstructural Protein, Helicase Activity, and Viral Replication by a Recombinant Human Antibody Clone

Hepatitis C virus (HCV) nonstructural protein 3 (NS3), with its protease, helicase, and NTPase enzymatic activities, plays a crucial role in viral replication, and therefore represents an ideal target for the development of anti-viral agents. We have developed a recombinant human antibody (Fab) that reacts with the helicase domain of HCV NS3. The affinity-purified Fab antibody completely inhibited the helicase activity of HCV NS3 at equimolar concentration. To evaluate the effect of the Fab on HCV replication, the clone encoding the Fab gene was put into an expression vector, which converts Fab into a complete IgG1 antibody. Using a DNA-based transfection model, we demonstrated that intracellular expression of this antibody resulted in significant reduction of HCV-negative strand RNA synthesis. Intracellular expression of this antibody into either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. These results support the use of recombinant antibody fragments to inhibit NS3 enzyme as a novel, feasible, and effective approach for inhibiting HCV replication.

Intracytoplasmic stable expression of IgG1 antibody targeting NS3 helicase inhibits replication of highly efficient hepatitis C Virus 2a clone

BackgroundHepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.ResultsThe antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.ConclusionRecombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.

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.

Altered Immune Responses in Rhesus Macaques Co-Infected with SIV and Plasmodium cynomolgi: An Animal Model for Coincident AIDS and Relapsing Malaria

Background Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens. Methodology/Principal Findings Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response. Conclusions/Significance These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

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.