scholarly journals Hepatitis C virus NS5A: tales of a promiscuous protein

2004 ◽  
Vol 85 (9) ◽  
pp. 2485-2502 ◽  
Author(s):  
Andrew Macdonald ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Replication ◽  
Viral Rna ◽  
Cell Physiology ◽  
Subgenomic Replicon ◽  
Cellular Environment ◽  
Cellular Context

The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation.

scholarly journals Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽  
2002 ◽  
Vol 297 (2) ◽  
pp. 298-306 ◽  
Author(s):  
I.Wayne Cheney ◽  
Suhaila Naim ◽  
Vicky C.H. Lai ◽  
Shannon Dempsey ◽  
Daniel Bellows ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Enzymatic Activity ◽  
Rna Replication ◽  
Viral Rna ◽  
Subgenomic Replicon ◽  
Replicon Cell ◽  
Ns5b Polymerase

scholarly journals Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

2004 ◽  
Vol 48 (12) ◽  
pp. 4784-4792 ◽  
Author(s):  
Kai Lin ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adverse Effects ◽  
Protease Inhibitor ◽  
Standard Of Care ◽  
Rna Replication ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

scholarly journals Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

2014 ◽  
Vol 58 (9) ◽  
pp. 5386-5394 ◽  
Author(s):  
Constance N. Wose Kinge ◽  
Christine Espiritu ◽  
Nishi Prabdial-Sing ◽  
Nomathamsaqa Patricia Sithebe ◽  
Mohsan Saeed ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Antiviral Agents ◽  
Rna Replication ◽  
Firefly Luciferase ◽  
Hcv Rna ◽  
Virus Genotype ◽  
Subgenomic Replicon

ABSTRACTHepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy.In vitroreplication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established anin vitroreplication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

scholarly journals The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

2008 ◽  
Vol 83 (5) ◽  
pp. 2163-2177 ◽  
Author(s):  
Daniel M. Jones ◽  
Arvind H. Patel ◽  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Rna Replication ◽  
Viral Rna ◽  
Focus Formation ◽  
Er Membrane

ABSTRACT Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.

scholarly journals Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

2020 ◽  
Vol 295 (35) ◽  
pp. 12426-12436 ◽  
Author(s):  
Lorillee Tallorin ◽  
Valerie A. Villareal ◽  
Chih-Yun Hsia ◽  
Mary A. Rodgers ◽  
Dominique J. Burri ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Rna Replication ◽  
Hcv Rna ◽  
Genome Replication ◽  
Subgenomic Replicon ◽  
Lipid Environment

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

scholarly journals Efficacy of a Peruvian Botanical Remedy (Sabell A4+) for Treating Liver Disease and Protecting Gastric Mucosal Integrity

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Mark G. Swain ◽  
John L. Wallace ◽  
D. Lorne Tyrrell ◽  
José Cabanillas ◽  
Steven K. H. Aung ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Gastric Mucosa ◽  
Virus Replication ◽  
Positive Interactions ◽  
Mucosal Integrity ◽  
Immune Mediated ◽  
Anti Inflammatory

The purpose of this study was to determine the efficacy of a Peruvian botanical formulation for treating disorders of hepatic function and gastric mucosal integrity. The formulation A4+ (Sabell Corporation) contains extracts of Curcuma longa rhizome, Cordia lutea flower, and Annona muricata leaf. Individually these plants have been used as traditional remedies for liver disease. We report the efficacy of A4+ and its components using a variety of in vitro and in vivo disease models. The methods used included tests for antioxidant, anti-inflammatory, and antiviral activity as well as mouse models of liver disease, including Concanavalin A-induced immune-mediated hepatitis and a bile duct ligation model for evaluating sickness behaviour associated with liver disease. Rat models were used to evaluate the gastric mucosal protective property of A4+ following indomethacin challenge and to evaluate its anti-inflammatory action in an “air pouch” model. In all tests, A4+ proved to be more effective than placebo. A4+ was antioxidant and anti-inflammatory and diminished Hepatitis C virus replication in vitro. In animal models, A4+ was shown to protect the liver from immune-mediated hepatitis, improve behavioural function in animals with late stage liver disease, and protect the rat gastric mucosa from ulceration following NSAID exposure. We conclude that A4+ ameliorated many aspects of liver injury, inhibited hepatitis C virus replication, and protected the gastric mucosa from NSAIDs. These varied beneficial properties appear to result from positive interactions between the three constituent herbs.

scholarly journals Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

2010 ◽  
Vol 285 (36) ◽  
pp. 28183-28190 ◽  
Author(s):  
Yin-Ju Chen ◽  
Yu-Hsuan Chen ◽  
Lu-Ping Chow ◽  
Ya-Hui Tsai ◽  
Pei-Hong Chen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Heat Shock Protein 72 ◽  
Replicase Complex

scholarly journals Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

2005 ◽  
Vol 86 (11) ◽  
pp. 3075-3080 ◽  
Author(s):  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication Assay ◽  
Cell Extracts ◽  
Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

scholarly journals mTORC1 Restricts Hepatitis C Virus Replication Through ULK1-mediated Suppression of miR-122 and Facilitates Post-replication Events

2018 ◽  
Author(s):  
Manish Kumar Johri ◽  
Hiren Vasantrai Lashkari ◽  
Dhiviya Vedagiri ◽  
Divya Gupta ◽  
Krishnan Harinivas Harshan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infection ◽  
Virus Replication ◽  
Viral Rna ◽  
Hcv Rna ◽  
Significant Drop ◽  
Mechanistic Target Of Rapamycin ◽  
Mtorc1 Activation ◽  
Rna Levels

ABSTRACTMechanistic target of rapamycin (mTOR) is an important kinase that assimilates several upstream signals including viral infection and facilitates appropriate response by the cell through two unique complexes mTORC1 and mTORC2. Here, we demonstrate that mTORC1 is activated early during HCV infection as antiviral response. Pharmacological inhibition of mTORC1 promoted HCV replication as suggested by elevated levels of HCV (+) and (-) RNA strands. This was accompanied by significant drop in extracellular HCV RNA levels indicating defective post-replication stages. The increase in viral RNA levels failed to augment intracellular infectious virion levels, suggesting that mTORC1 inhibition is detrimental to post-replication steps. Lower infectivity of the supernatant confirmed this observation. Depletion of Raptor and ULK1 accurately reproduced these results suggesting that mTORC1 imparted these effects on HCV through mTORC1-ULK1 arm. Interestingly, ULK1 depletion resulted in increased levels of miR-122, a critical host factor for HCV replication, thus revealing a new mechanism of regulation by ULK1. The binary effect of mTORC1 on HCV replication and egress suggests that mTORC1-ULK1 could be critical in replication: egress balance. Interestingly we discover that ULK1 depletion did not interfere with autophagy in Huh7.5 cells and hence the effects on HCV replication and post-replication events are not resultant of involvement of autophagy. Our studies demonstrate an overall ULK1 mediated anti-HCV function of mTORC1 and identifies an ULK1-independent autophagy that allows HCV replication in spite of mTORC1 activation.

Sign in / Sign up

Export Citation Format

Share Document