Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

Fridell, Robert A.; Qiu, Dike; Valera, Lourdes; Wang, Chunfu; Rose, Ronald E.; Gao, Min

doi:10.1128/jvi.00253-11

Cited by 133 publications

(130 citation statements)

References 60 publications

(116 reference statements)

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“…Transient replication assays were performed as previously described (26,29). Briefly, replicon transcripts, generated with a Ribomax T7 express system (Promega Corp., Madison, WI) from XbaIlinearized plasmids, were transfected into Huh7.5 cells by using DMRIE-C (liposome formulation of the cationic lipid DMRIE [1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide] and cholesterol) reagent (Invitrogen, Corp., Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Human hepatoma (Huh-7.5) and baby hamster kidney (BHK1) cells were maintained as previously described (29). A plasmid encoding a bicistronic replicon with the Con1 HCV 5= untranslated region (5=UTR) and internal ribosomal entry site (IRES) sequences, a Renilla luciferase (Rluc) reporter gene, and an encephalomyocarditis virus (EMCV) IRES followed by the NS3 through 3=UTR region from the JFH1 strain has been described (26). NS5A amino acid substitutions were introduced into the replicon plasmid by replacing a BspE1-BspE1 fragment with PCR-generated fragments and were confirmed by sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

“…As a consequence of the membrane compartmentalization and sequestration of viral replication complexes, diffusion of virus-encoded replication proteins between replication complexes encoded by different viral genomes may be limited, leading to a preference for cis-dominant replication functions. For HCV, cis-acting RNA replication functions have been reported for NS3, NS4A, and NS5B, while transacting functions have been reported for NS5A and NS4B (25)(26)(27)(28).…”

Section: H Epatitis C Virus (Hcv) Is An Enveloped Positive-sense Singmentioning

confidence: 99%

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Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Fridell

Valera

Qiu

et al. 2013

J Virol

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Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypoand hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication. H epatitis C virus (HCV) is an enveloped positive-sense singlestranded RNA virus of the genus Hepacivirus in the familyFlaviviridae. The ϳ9.6-kb HCV genome encodes an ϳ3,000-amino-acid (aa) polyprotein that is cleaved by viral and cellular proteases into 10 mature proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed in references 1 and 2). Core is the viral capsid protein, while E1 and E2 are highly glycosylated envelope proteins important for viral entry. P7, a small transmembrane ion channel protein, and NS2, a membrane-associated protease, are required for assembly and release of infectious virions. The remaining nonstructural (NS) proteins are essential components of the HCV RNA replication complex. NS5B is an RNAdependent RNA polymerase that catalyzes synthesis of positiveand negative-strand RNA. NS3 and its cofactor NS4A function as a serine protease responsible for releasing mature NS proteins from the polyprotein precursor. NS3 also possesses RNA helicase and NTPase activities essential for RNA replication. NS4B is an integral membrane protein likely involved in formation of intracellular membranous compartments where viral replication occurs. The final viral component of the replication c...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: H Epatitis C Virus (Hcv) Is An Enveloped Positive-sense Singmentioning

confidence: 99%

See 1 more Smart Citation

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Fridell

Valera

Qiu

et al. 2013

J Virol

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“…12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors. [12][13][14] Furthermore, NS5A inhibitors have been shown to induce redistribution of NS5A from endoplasmic reticulum-derived foci, [12][13][14] possibly to lipid droplets, 12 and limit hyperphosphorylation of NS5A, [14][15][16] although these effects may be indirect. Finally, it has been suggested that NS5A inhibitors may disrupt interactions of NS5A with HCV RNA, other viral proteins, and/or host factors that are coopted by NS5A during the HCV life cycle.…”

Section: Hcv Ns5a Inhibitors Disrupt Replication Factory Formation: Amentioning

confidence: 99%

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

Eyre

Beard

2014

Gastroenterology

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After the embargo period via non-commercial hosting platforms such as their institutional repository  via commercial sites with which Elsevier has an agreement In all cases accepted manuscripts should: link to the formal publication via its DOI  bear a CC-BY-NC-ND license -this is easy to do, click here to find out how  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy  not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article Embargo ISSN Journal Name Embargo Period (months)0016-5085 Gastroenterology

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“…With the promising results of a recent Phase I clinical trial for the NS5A-targeting BMS-790052 compound against HCV infection in patients who followed a once-daily, dosing regimen [119], there is huge potential for developing additional inhibition strategies against the NS5A protein. Indeed, the amphipathic character of the AH is conserved across all HCV isolates to date [63,64,67], making the AH far less likely to evolve resistance mutations [120,121]. The QCM-D vesicle rupturing assay could be alternatively used to screen chemical libraries for small molecules that inhibit vesicle rupturing by the NS5A AH peptide.…”

Section: Outlook On Engineering Strategies For Antiviral Drug Developmentioning

confidence: 99%

Model Membrane Platforms for Biomedicine: Case Study on Antiviral Drug Development

Jackman

Cho

2012

Biointerphases

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As one of the most important interfaces in cellular systems, biological membranes have essential functions in many activities such as cellular protection and signaling. Beyond their direct functions, they also serve as scaffolds to support the association of proteins involved in structural support, adhesion, and transport. Unfortunately, biological processes sometimes malfunction and require therapeutic intervention. For those processes which occur within or upon membranes, it is oftentimes difficult to study the mechanism in a biologically relevant, membranous environment. Therefore, the identification of direct therapeutic targets is challenging. In order to overcome this barrier, engineering strategies offer a new approach to interrogate biological activities at membrane interfaces by analyzing them through the principles of the interfacial sciences. Since membranes are complex biological interfaces, the development of simplified model systems which mimic important properties of membranes can enable fundamental characterization of interaction parameters for such processes. We have selected the hepatitis C virus (HCV) as a model viral pathogen to demonstrate how model membrane platforms can aid antiviral drug discovery and development. Responsible for generating the genomic diversity that makes treating HCV infection so difficult, viral replication represents an ideal step in the virus life cycle for therapeutic intervention. To target HCV genome replication, the interaction of viral proteins with model membrane platforms has served as a useful strategy for target identification and characterization. In this review article, we demonstrate how engineering approaches have led to the discovery of a new functional activity encoded within the HCV nonstructural 5A protein. Specifically, its N-terminal amphipathic, α-helix (AH) can rupture lipid vesicles in a size-dependent manner. While this activity has a number of exciting biotechnology and biomedical applications, arguably the most promising one is in antiviral medicine. Based on the similarities between lipid vesicles and the lipid envelopes of virus particles, experimental findings from model membrane platforms led to the prediction that a range of medically important viruses might be susceptible to rupturing treatment with synthetic AH peptide. This hypothesis was tested and validated by molecular virology studies. Broad-spectrum antiviral activity of the AH peptide has been identified against HCV, HIV, herpes simplex virus, and dengue virus, and many more deadly pathogens. As a result, the AH peptide is the first in class of broad-spectrum, lipid envelope-rupturing antiviral agents, and has entered the drug pipeline. In summary, engineering strategies break down complex biological systems into simplified biomimetic models that recapitulate the most important parameters. This approach is particularly advantageous for membrane-associated biological processes because model membrane platforms provide more direct characterization of target interactions than is possible with other methods. Consequently, model membrane platforms hold great promise for solving important biomedical problems and speeding up the translation of biological knowledge into clinical applications.

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Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

Cited by 133 publications

References 60 publications

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

Model Membrane Platforms for Biomedicine: Case Study on Antiviral Drug Development

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