scholarly journals Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences

2002 ◽  
Vol 76 (19) ◽  
pp. 9563-9574 ◽  
Author(s):  
Robert M. Smith ◽  
Cherie M. Walton ◽  
Catherine H. Wu ◽  
George Y. Wu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Secondary Structure ◽  
Rna Structure ◽  
Genome Stability ◽  
Rnase H ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.

scholarly journals Quantitative Analysis of the Hepatitis C Virus Replication Complex

2005 ◽  
Vol 79 (21) ◽  
pp. 13594-13605 ◽  
Author(s):  
Doris Quinkert ◽  
Ralf Bartenschlager ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Viral Proteins ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

scholarly journals Effects of Mutations of the Initiation Nucleotides on Hepatitis C Virus RNA Replication in the Cell

2004 ◽  
Vol 78 (7) ◽  
pp. 3633-3643 ◽  
Author(s):  
Zhaohui Cai ◽  
T. Jake Liang ◽  
Guangxiang Luo
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Rna Viruses ◽  
Rna Replication ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
Huh7 Cells ◽  
Cell Colony

ABSTRACT Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5′ and 3′ ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5′ end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5′ end dramatically reduced the efficiency of cell colony formation. Likewise, the 3′-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3′ end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5′-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5′ end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5′-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3′-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3′ ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3′ end. We also determined the 5′-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5′ end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

2012 ◽  
Vol 57 (1) ◽  
pp. 436-444 ◽  
Author(s):  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Izuru Ando ◽  
Kunihiro Hirahara ◽  
Tsutomu Shibata ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Resistance ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
Resistance Mutations ◽  
In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

scholarly journals Cellular RNA Helicase p68 Relocalization and Interaction with the Hepatitis C Virus (HCV) NS5B Protein and the Potential Role of p68 in HCV RNA Replication

2004 ◽  
Vol 78 (10) ◽  
pp. 5288-5298 ◽  
Author(s):  
Phuay-Yee Goh ◽  
Yee-Joo Tan ◽  
Siew Pheng Lim ◽  
Y. H. Tan ◽  
Seng Gee Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Line ◽  
Rna Helicase ◽  
Hcv Rna ◽  
Activity Levels ◽  
Nonstructural Proteins ◽  
Negative Strand ◽  
Replicon Cell Line ◽  
Rna Helicase P68

ABSTRACT Chronic infection by hepatitis C virus (HCV) can lead to severe hepatitis and cirrhosis and is closely associated with hepatocellular carcinoma. The replication cycle of HCV is poorly understood but is likely to involve interaction with host factors. In this report, we show that NS5B, the HCV RNA-dependent RNA polymerase (RdRp), interacts with a human RNA helicase, p68. Transient expression of NS5B alone, as well as the stable expression of all the nonstructural proteins in a HCV replicon-bearing cell line (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113), causes the redistribution of endogenous p68 from the nucleus to the cytoplasm. Deletion of the C-terminal two-thirds of NS5B (NS5BΔC) dramatically reduces its coimmunoprecipitation (co-IP) with endogenous p68, while the deletion of the N-terminal region (NS5BΔN1 and NS5BΔN2) does not affect its interaction with p68. In consistency with the co-IP results, NS5BΔC does not cause the relocalization of p68 whereas NS5BΔN1 does. With a replicon cell line, we were not able to detect a change in positive- and negative-strand synthesis when p68 levels were reduced using small interfering RNA (siRNA). In cells transiently transfected with a full-length HCV construct, however, the depletion (using specific p68 siRNA) of endogenous p68 correlated with a reduction in the transcription of negative-strand from positive-strand HCV RNA. Overexpression of NS5B and NS5BΔN1, but not that of NS5BΔC, causes a reduction in the negative-strand synthesis, indicating that overexpressed NS5B and NS5BΔN1 sequesters p68 from the replication complexes (thus reducing their replication activity levels). Identification of p68 as a cellular factor involved in HCV replication, at least for cells transiently transfected with a HCV expression construct, is a step towards understanding HCV replication.

scholarly journals Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

2021 ◽  
Author(s):  
Mamata Panigrahi ◽  
Michael Palmer ◽  
Joyce A Wilson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Structure ◽  
Genome Stability ◽  
Internal Ribosomal Entry Site ◽  
Terminal Region ◽  
Translation Regulation ◽  
Virus Propagation ◽  
Entry Site

The 5’UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5’ terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5’ terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5’ UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5’ UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5’ UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle.

scholarly journals Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus

2018 ◽  
Vol 19 (9) ◽  
pp. 2771 ◽  
Author(s):  
Yoo Cho ◽  
Hwan Lee ◽  
Hyojeung Kang ◽  
Hyosun Cho
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nk Cells ◽  
Natural Killer ◽  
Core Protein ◽  
Lymphoid Cells ◽  
Hcv Rna ◽  
Cell Infection ◽  
Hcv Core Protein

HCV genotype 2a strain JFH-1 replicates and produces viral particles efficiently in human hepatocellular carcinoma (huh) 7.5 cells, which provide a stable in vitro cell infection system for the hepatitis C virus (HCVcc system). Natural killer (NK) cells are large lymphoid cells that recognize and kill virus-infected cells. In this study, we investigated the interaction between NK cells and the HCVcc system. IL-10 is a typical immune regulatory cytokine that is produced mostly by NK cells and macrophages. IL-21 is one of the main cytokines that stimulate the activation of NK cells. First, we used anti-IL-10 to neutralize IL-10 in a coculture of NK cells and HCVcc. Anti-IL-10 treatment increased the maturation of NK cells by enhancing the frequency of the CD56+dim population in NK-92 cells. However, with anti-IL-10 treatment of NK cells in coculture with J6/JFH-1-huh 7.5 cells, there was a significant decrease in the expression of STAT1 and STAT5 proteins in NK-92 cells and an increase in the HCV Core and NS3 proteins. In addition, rIL-21 treatment increased the frequency of the CD56+dim population in NK-92 cells, Also, there was a dramatic increase in the expression of STAT1 and STAT5 proteins in rIL-21 pre-stimulated NK cells and a decrease in the expression of HCV Core protein in coculture with J6/JFH-1-huh 7.5 cells. In summary, we found that the functional activation of NK cells can be modulated by anti-IL-10 or rIL-21, which controls the expression of HCV proteins as well as HCV RNA replication.

scholarly journals 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

2008 ◽  
Vol 52 (6) ◽  
pp. 2097-2110 ◽  
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.

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

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
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.

scholarly journals Modulation of alpha interferon anti-hepatitis C virus activity by ISG15

2009 ◽  
Vol 90 (12) ◽  
pp. 2929-2939 ◽  
Author(s):  
Pong Kian Chua ◽  
Matthew F. McCown ◽  
Sonal Rajyaguru ◽  
Simran Kular ◽  
Ram Varma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Effect ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Moderate Increase ◽  
Knock Down ◽  
Inducible Genes

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-α) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-α, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-α-specific effect, which was not observed with IFN-γ, correlated with an increase in expression of the IFN-α-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-α-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-α after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-α-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-α treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-α pathway and its antiviral effect.

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