Protease Inhibitor–Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus

Shimakami, Tetsuro; Welsch, Christoph; Yamane, Daisuke; McGivern, David R.; Yi, Min Kyung; Zeuzem, Stefan; Lemon, Stanley M.

doi:10.1053/j.gastro.2010.10.056

Cited by 128 publications

(212 citation statements)

References 28 publications

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“…Replication was detected by expression of a Gaussia princeps (GLuc) luciferase gene inserted in the HCV ORF distant from the structures of interest (Fig. 4A) following transfection of synthetic transcripts into Huh-7.5 cells (37). GLuc activity reflects viral polyprotein synthesis and is a good surrogate measure of genome replication.…”

Section: Resultsmentioning

confidence: 99%

“…We also measured production of infectious virus in assays that depend on competency at every stage of the viral replication cycle (37,38). Mutant J750 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Structure-disrupting mutations were created in the JFH1-QL and H77S.3 genomes and their GLuc2A counterparts (Table S2), and synthetic RNAs produced from these clones were then assayed for their replication competence by measuring luciferase expression and infectious virus yields (36)(37)(38).…”

mentioning

confidence: 99%

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Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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119

188

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Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

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Section: Resultsmentioning

confidence: 99%

“…We also measured production of infectious virus in assays that depend on competency at every stage of the viral replication cycle (37,38). Mutant J750 (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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119

188

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“…The precise conservation of some NS3 protease segments implies that certain amino acid changes affect enzyme viability. Indeed, mutations associated with drug resistance and CTL escape have been shown to have an impact on HCV fitness (44). Yet, little is known about the distribution of catalytic efficiency among NS3 protease mutations at the population level.…”

Section: Discussionmentioning

confidence: 99%

Complexity and Catalytic Efficiency of Hepatitis C Virus (HCV) NS3 and NS4A Protease Quasispecies Influence Responsiveness to Treatment with Pegylated Interferon plus Ribavirin in HCV/HIV-Coinfected Patients

Aparicio

Franco

Parera

et al. 2011

J Virol

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Hepatitis C virus (HCV) nonstructural protein 3 (NS3) contains a serine protease that cleaves the virus-encoded polyprotein and inactivates cellular proteins required for innate immunity. HCV NS3/4A protease functions as an antagonist of virus-induced interferon (IFN) regulatory factor 3 activation and IFN-␤ expression through its ability to block retinoic acid-inducible gen I (RIG-I) and Toll-like receptor 3 signaling by cleaving caspase recruitment domain adaptor-inducing IFN-␤ (Cardif) and Toll/interleukin-1 (IL-1) receptor domain-containing adaptor-inducing IFN proteins, respectively. NS3/4A protease activity allows the virus to evade the cellular innate immune response, which may influence the subsequent development of adaptive immunity to HCV, virus persistence, and the response to IFNbased therapy (21).HCV is the causal agent of chronic liver infection, which afflicts more than 170 million people worldwide

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“…HeLa cells were electroporated at 300 V, 500 μF, and ∞ Ω, and MEFs at 400 V, 250 μF, and ∞ Ω. Cells were harvested at intervals and supernatant fluids assayed for GLuc activity (31) and HCV RNA abundance in cell lysates assessed by Northern blotting (11). Polyadenylated reporter RNAs encoding firefly or Cypridina luciferase were cotransfected to monitor transfection efficiency (11).…”

Section: Methodsmentioning

confidence: 99%

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

Shimakami

Yamane

Jangra

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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351

380

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MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

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Protease Inhibitor–Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus

Cited by 128 publications

References 28 publications

Functionally conserved architecture of hepatitis C virus RNA genomes

Functionally conserved architecture of hepatitis C virus RNA genomes

Complexity and Catalytic Efficiency of Hepatitis C Virus (HCV) NS3 and NS4A Protease Quasispecies Influence Responsiveness to Treatment with Pegylated Interferon plus Ribavirin in HCV/HIV-Coinfected Patients

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

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