Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Computer analysis of 158 hepatitis C virus (HCV) 5′ untranslated region (5′ UTR) sequences from the six genotypes showed that the 5′ UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5′ UTR of the plus-strand RNA and the 3′ end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

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Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3′-end of virus minus-strand RNA

FEBS Journal ◽

10.1111/j.1742-4658.2005.04804.x ◽

2005 ◽

Vol 272 (15) ◽

pp. 3872-3886 ◽

Cited By ~ 16

Author(s):

Thérèse Astier-Gin ◽

Pantxika Bellecave ◽

Simon Litvak ◽

Michel Ventura

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Minus Strand ◽

Ns5b Polymerase

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An oligonucleotide complementary to the SL-B1 domain in the 3′-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

Virology ◽

10.1016/s0042-6822(03)00393-3 ◽

2003 ◽

Vol 314 (1) ◽

pp. 206-220 ◽

Cited By ~ 3

Author(s):

Sandrine Reigadas ◽

Michel Ventura ◽

Marie-Line Andreola ◽

Justine Michel ◽

Sergei Gryaznov ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Minus Strand ◽

Viral Polymerase ◽

B1 Domain

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Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

Journal of Virology ◽

10.1128/jvi.77.9.5401-5414.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5401-5414 ◽

Cited By ~ 131

Author(s):

Maria Dimitrova ◽

Isabelle Imbert ◽

Marie Paule Kieny ◽

Catherine Schuster

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interactions ◽

Laser Scanning ◽

Ex Vivo ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

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‘Distribution of Hepatitis C Virus genotypes in northern Greece in the last decade: descriptive analysis and clinical correlations’

Global Health Epidemiology and Genomics ◽

10.1017/gheg.2019.4 ◽

2019 ◽

Vol 4 ◽

Author(s):

G. Gioula ◽

E. Sinakos ◽

E. Gigi ◽

I. Goulis ◽

T. Vasiliadis ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Drug Use ◽

Intravenous Drug ◽

Genotype Distribution ◽

Intravenous Drug Use ◽

Genotype 1 ◽

Genotype 3 ◽

Northern Greece ◽

Hcv Genotype

Abstract Hepatitis C virus (HCV) represents a major public health problem, while the identification of a HCV genotype is clinically very important for therapy prescription. The aim of the present study was to determine the HCV genotype distribution patients from northern Greece with HCV RNA positive viral load and to identify whether there is a shift in this distribution, during 2009–2017. The study was performed on 915 HCV positive patients and according to the results, genotype 3 was the most prevalent genotype (Ν = 395, 43.2%) followed by genotype 1 (Ν = 361, 39.5%). Regarding the gender of the patients, genotype 1 was mostly detected in women. Moreover, genotype 1 was associated with higher viral loads, while genotype 3 was most frequently detected in patients with a history of intravenous drug use. In conclusion, our results show that genotype 3 is the most prevalent genotype in Greece during the last decade as opposed to older epidemiological studies, likely due to intravenous drug use becoming the major source of infection.

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The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model

Gut ◽

10.1136/gut.2006.108647 ◽

2007 ◽

Vol 56 (9) ◽

pp. 1302-1308 ◽

Cited By ~ 68

Author(s):

C Hourioux ◽

R Patient ◽

A Morin ◽

E Blanchard ◽

A Moreau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Cellular Model ◽

Genotype 3 ◽

Virus Core

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The impact of universal access to DAA and real-world treatment outcome amongst genotype 3 hepatitis C virus-infected prisoners

Journal of Hepatology ◽

10.1016/s0168-8278(20)31128-4 ◽

2020 ◽

Vol 73 ◽

pp. S313

Author(s):

Yu Jun Wong ◽

Fria May Gloriba Manejero ◽

Kim Wei Lim ◽

Sin Yoong Chong ◽

Linn War Mai ◽

...

Keyword(s):

Treatment Outcome ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Real World ◽

Universal Access ◽

Genotype 3 ◽

The Impact

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Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01227-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2097-2110 ◽

Cited By ~ 22

Author(s):

Pantxika Bellecave ◽

Christian Cazenave ◽

Julie Rumi ◽

Cathy Staedel ◽

Ophélie Cosnefroy ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Selection Procedure ◽

Inhibitory Effect ◽

Hcv Rna ◽

Dna Aptamers ◽

Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

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Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

Journal of Virology ◽

10.1128/jvi.00032-07 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5270-5283 ◽

Cited By ~ 80

Author(s):

Marco Binder ◽

Doris Quinkert ◽

Olga Bochkarova ◽

Rahel Klein ◽

Nikolina Kezmic ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Negative Strand ◽

Virus Rna ◽

Nontranslated Region ◽

Genotype 2 ◽

Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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