Hepatitis C Viral Replication Complex

The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.

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An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

Journal of Virology ◽

10.1128/jvi.78.20.11393-11400.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11393-11400 ◽

Cited By ~ 112

Author(s):

Menashe Elazar ◽

Ping Liu ◽

Charles M. Rice ◽

Jeffrey S. Glenn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hcv Rna ◽

Membrane Association ◽

Amphipathic Helix ◽

Nonstructural Proteins ◽

Replication Complex ◽

Cytoplasmic Membranes ◽

Positive Strand Rna

ABSTRACT Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

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Inhibitory Effects of Amentoflavone and Orobol on Daclatasvir-Induced Resistance-Associated Variants of Hepatitis C Virus

The American Journal of Chinese Medicine ◽

10.1142/s0192415x18500441 ◽

2018 ◽

Vol 46 (04) ◽

pp. 835-852 ◽

Cited By ~ 5

Author(s):

Wei-Ping Lee ◽

Keng-Li Lan ◽

Shi-Xian Liao ◽

Yi-Hsiang Huang ◽

Ming-Chih Hou ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Entry ◽

Antiviral Agents ◽

Cost Effective ◽

Pegylated Interferon ◽

Chinese Herbs ◽

Inhibitory Effects ◽

Direct Acting Antiviral ◽

Direct Acting

Hepatitis C virus (HCV) is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite rapid progress in the development of direct-acting antivirals (DAA) against HCV infection in recent years, cost-effective antiviral drugs with more affordable prices still need to be developed. In this study, we screened a library of natural compounds to identify natural HCV inhibitors. The library of the pure compounds extracted from Chinese herbs deposited in the chemical bank of National Research Institute of Chinese Medicine (NRICM), Taiwan was screened in the cell culture-derived HCV (HCVcc) system. We identified the flavone or flavan-based compounds amentoflavone, 7,4[Formula: see text]-dihydroxyflavanone, and orobol with the inhibition of viral entry, replication, and translation of the HCV life cycle. Amentoflavone and orobol also showed inhibitory effects on resistant-associated variants to the NS5A inhibitor daclatasvir. The results of this study have the potential to benefit patients who are intolerant to the adverse effect of pegylated interferon or who harbor resistant strains refractory to treatment by current direct-acting antiviral agents.

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Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

RNA ◽

10.1261/rna.2144203 ◽

2003 ◽

Vol 9 (3) ◽

pp. 331-345 ◽

Cited By ~ 66

Author(s):

M. YI

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Structure Function ◽

Function Analysis ◽

Rna Replication ◽

Viral Rna ◽

Genomic Rna ◽

Stem Loop

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Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons

FEBS Journal ◽

10.1111/j.1742-4658.2005.04986.x ◽

2005 ◽

Vol 272 (22) ◽

pp. 5910-5922 ◽

Cited By ~ 35

Author(s):

Dominik Jarczak ◽

Mortimer Korf ◽

Carmela Beger ◽

Michael P. Manns ◽

Martin Krüger

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Translation ◽

Rna Replication ◽

Virus Sequence ◽

Hairpin Ribozymes

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Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A

Journal of Virology ◽

10.1128/jvi.79.2.896-909.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 896-909 ◽

Cited By ~ 71

Author(s):

Nicole Appel ◽

Ulrike Herian ◽

Ralf Bartenschlager

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Functional Organization ◽

Nonstructural Protein ◽

Rna Replication ◽

Hcv Rna ◽

Replication Complex ◽

Diarrhea Virus ◽

Amino Terminal ◽

Essential Components

ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

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Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

Journal of Virology ◽

10.1128/jvi.78.7.3480-3488.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3480-3488 ◽

Cited By ~ 247

Author(s):

Lu Gao ◽

Hideki Aizaki ◽

Jian-Wen He ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Raft ◽

Critical Role ◽

Rna Replication ◽

Dominant Negative ◽

Host Protein ◽

Hcv Rna ◽

Replication Complex ◽

Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

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Genetic Interactions between Hepatitis C Virus Replicons

Journal of Virology ◽

10.1128/jvi.78.21.12085-12089.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 12085-12089 ◽

Cited By ~ 28

Author(s):

Matthew J. Evans ◽

Charles M. Rice ◽

Stephen P. Goff

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Genetic Interactions ◽

Hcv Rna ◽

Replication Complex ◽

Huh7 Cells ◽

High Level

ABSTRACT To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

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The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

Journal of Virology ◽

10.1128/jvi.73.1.307-315.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 307-315 ◽

Cited By ~ 33

Author(s):

Sean P. J. Whelan ◽

Gail W. Wertz

Keyword(s):

Vesicular Stomatitis Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Genomic Rna ◽

Wild Type ◽

Rna Sequences ◽

Defective Interfering Particles ◽

Cis Acting ◽

Efficient Replication ◽

Vesicular Stomatitis

ABSTRACT The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that acis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.

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Dehydrojuncusol, a Natural Phenanthrene Compound Extracted fromJuncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.02009-18 ◽

2019 ◽

Vol 93 (10) ◽

Cited By ~ 4

Author(s):

Marie-Emmanuelle Sahuc ◽

Ramla Sahli ◽

Céline Rivière ◽

Véronique Pène ◽

Muriel Lavie ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Treatment Failure ◽

Natural Compound ◽

Rna Replication ◽

Direct Acting Antivirals ◽

Ns5a Protein ◽

Juncus Maritimus ◽

Direct Acting

ABSTRACTRecent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract ofJuncus maritimusLam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. Noin vitrotoxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted fromJ. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCETens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated fromJuncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

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