scholarly journals Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 17 ◽  
Author(s):  
Elodie Rance ◽  
Jerome Tanner ◽  
Caroline Alfieri
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Progeny Virus ◽  
Hcv Rna ◽  
Genomic Rna ◽  
Coding Region ◽  
Rna Dependent Rna Polymerase ◽  
High Affinity

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication.

scholarly journals The Octadecyloxyethyl Ester of (S)-9-[3-Hydroxy-2-(Phosphonomethoxy) Propyl]Adenine Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication in Genotype 1A, 1B, and 2A Replicons

2009 ◽  
Vol 53 (6) ◽  
pp. 2660-2662 ◽  
Author(s):  
David L. Wyles ◽  
Kelly A. Kaihara ◽  
Brent E. Korba ◽  
Robert T. Schooley ◽  
James R. Beadle ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Active Compound ◽  
Hcv Rna ◽  
Genotype 1A ◽  
B Virus ◽  
Immunodeficiency Virus

ABSTRACT The octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of orthopoxvirus, herpesvirus, human immunodeficiency virus type 1, and hepatitis B virus replication in vitro. HDP and ODE esters of (S)-HPMPA and (R)-HPMPA were evaluated for their activity in hepatitis C virus (HCV) replicon assays using luciferase (1B and 2A replicons) or RNA (1B) quantification. The ODE ester of (S)-HPMPA [ODE-(S)-HPMPA] was the most active compound, with 50% effective concentrations (EC50s) in the 0.69 to 1.31 μM range. HDP and ODE esters of (R)-HPMPA were severalfold less active, while (S)-HPMPA and (R)-HPMPA were inactive. In genotype 1A and 1B replicons analyzed by HCV RNA analysis, ODE-(S)-HPMPA was the most active compound, with EC50s of 1.8 and 2.1 μM, respectively.

scholarly journals Characterization of Hepatitis C Virus Subgenomic Replicon Resistance to Cyclosporine In Vitro

2007 ◽  
Vol 81 (11) ◽  
pp. 5829-5840 ◽  
Author(s):  
John M. Robida ◽  
Heather B. Nelson ◽  
Zhe Liu ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cellular Protein ◽  
Live Cells ◽  
Hcv Rna ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Cyclophilin B

ABSTRACT Treatment of hepatitis C virus (HCV) infection has been met with less than satisfactory results due primarily to its resistance to and significant side effects from alpha interferon (IFN-α). New classes of safe and broadly acting treatments are urgently needed. Cyclosporine (CsA), an immunosuppressive and anti-inflammatory drug for organ transplant patients, has recently been shown to be highly effective in suppressing HCV replication through a mechanism that is distinct from the IFN pathway. Here we report the selection and characterization of HCV replicon cells that are resistant to CsA treatment in vitro, taking advantage of our ability to sort live cells that are actively replicating HCV RNA in the presence of drug treatments. This resistance is specific to CsA as the replicon cells most resistant to CsA were still sensitive to IFN-α and a polymerase inhibitor. We demonstrate that the resistant phenotype is not a result of general enhanced replication and, furthermore, that mutations in the coding region of HCV NS5B contribute to the resistance. Interestingly, a point mutation (I432V) isolated from the most resistant replicon was able to rescue a lethal mutation (P540A) in NS5B that disrupts its interaction with its cofactor, cyclophilin B (CypB), even though the I432V mutation is located outside of the reported CypB binding site (amino acids 520 to 591). Our results demonstrate that CsA exerts selective pressure on the HCV genome, leading to the emergence of resistance-conferring mutations in the viral genome despite acting upon a cellular protein.

scholarly journals Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

2003 ◽  
Vol 77 (7) ◽  
pp. 4149-4159 ◽  
Author(s):  
Lu Gao ◽  
Hong Tu ◽  
Stephanie T. Shi ◽  
Ki-Jeong Lee ◽  
Miyuki Asanaka ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon ◽  
C Terminus ◽  
Huh7 Cells

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

Monitoring hepatitis C virus (HCV) RNA-dependent RNA polymerase oligomerization by a FRET-based in vitro system

2010 ◽  
Vol 87 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Itxaso Bellón-Echeverría ◽  
Alberto José López-Jiménez ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
In Vitro System

scholarly journals Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

2020 ◽  
Author(s):  
Rosario Sabariegos ◽  
Ana M. Ortega-Prieto ◽  
Luis Díaz-Martínez ◽  
Ana Grande-Pérez ◽  
Isabel Gallego ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Polymerase Activity ◽  
Progeny Virus ◽  
Inhibition Mechanism ◽  
Hcv Rna ◽  
Template Switching ◽  
Human Hepatoma Cells

AbstractIn the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua and not the other standard nucleosides inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphate (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTP or NDP modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.Author summaryRibonucleoside metabolism is essential for replication of RNA viruses. In this article we describe the antiviral activity of the natural ribonucleoside guanosine (Gua). We demonstrate that hepatitis C virus (HCV) replication is inhibited in the presence of increasing concentrations of this ribonucleoside and that this inhibition does not occur as a consequence of a direct inhibition of HCV polymerase. Cells exposed to increasing concentrations of Gua show imbalances in the intracellular concentrations of nucleoside-diphosphates and triphosphates and as the virus is passaged in these cells, it accumulates mutations that reduce its infectivity and decimate its normal spreading capacity.

scholarly journals Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

2010 ◽  
Vol 84 (12) ◽  
pp. 5923-5935 ◽  
Author(s):  
S. Chinnaswamy ◽  
A. Murali ◽  
P. Li ◽  
K. Fujisaki ◽  
C. C. Kao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Rna Synthesis ◽  
De Novo ◽  
Enzyme Concentration ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.

scholarly journals Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA

1999 ◽  
Vol 80 (12) ◽  
pp. 3109-3113 ◽  
Author(s):  
Juraj Petrik ◽  
Hayley Parker ◽  
Graeme J. M. Alexander
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Middle Part ◽  
Hcv Rna ◽  
Binding Affinities ◽  
Genomic Rna ◽  
Coding Region ◽  
Multifunctional Protein ◽  
Polypyrimidine Tract Binding Protein ◽  
Replicase Complex

The unique poly(U/UC) tract, the middle part of the tripartite 3′ non-coding region (3′NCR) of hepatitis C virus (HCV) genomic RNA, may represent a recognition signal for the HCV replicase complex. In this study, several proteins binding specifically to immobilized ribooligonucleotide r(U)25 mimicking this structure were identified using cytosolic extracts from HCV-negative or -positive liver explants, and a prominent 36 kDa protein was studied further. Competition experiments including homoribopolymers revealed binding affinities in the order: oligo/poly(U)≫(A)≫(C)≫(G). The protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein known to bind RNA. GAPDH bound efficiently to the full-length HCV RNA and binding to various 3′NCR constructs revealed critical dependence upon the presence of the middle part of the 3′NCR. Polypyrimidine tract-binding protein, described previously to bind the 3′NCR, did not bind efficiently to the middle part of 3′NCR and was captured from liver extracts in considerably smaller quantities.

scholarly journals Dinucleotide Analogues as Novel Inhibitors of RNA-Dependent RNA Polymerase of Hepatitis C Virus

2003 ◽  
Vol 47 (8) ◽  
pp. 2674-2681 ◽  
Author(s):  
Weidong Zhong ◽  
Haoyun An ◽  
Dinesh Barawkar ◽  
Zhi Hong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Replication Initiation ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Initiation Process

ABSTRACT Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5′-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.

scholarly journals In Vitro Replication of HCV RNA in Peripheral Blood Mononuclear Cells Isolated from Patients Undergoing Treatment for Hepatitis C Virus Infection

2020 ◽  
Vol 20 (9) ◽  
Author(s):  
Golnoosh Zarnegar ◽  
Ali Farhadi ◽  
Noorossadat Seyyedi ◽  
Farzaneh Aboualizadeh ◽  
Reza Ranjbaran ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Mononuclear Cells ◽  
Lymphocyte Culture ◽  
Hcv Rna ◽  
Coding Region ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Background: Although the liver is the main site for the Hepatitis C Virus (HCV) replication, there is still an essential debate about extrahepatic HCV reservoirs. Objectives: It has been proposed that Peripheral Blood Mononuclear Cells (PBMCs) could be the possible virus replication sites. Therefore, PBMCs may be candidates for recurrent HCV infection after achieving Sustained Virologic Response (SVR). In this study, we designed a lymphocyte culture to explore more about virus replication in PBMCs collected from patients with chronic hepatitis C. Methods: Plasma and PBMC samples were collected from 16 randomly selected seropositive patients for the anti-HCV antibody. Four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. PBMCs were isolated from whole blood. Between 106-107 cells were cultured with optimized concentrations of IL-2 (10 mg/ml) and phytohemagglutinin A (5 mg/ml). Total RNA was extracted from the first collected sera and harvested lymphocytes. Constructed plasmids containing the NCR coding region were used to plot the standard curve for the relative quantification of SYBR green real-time PCR. The sensitivity and specificity of the detection were established by using plasmids containing cDNA. Results: With this plasmid containing the NCR coding region, the Limit of Detection (LOD) of in-house-developed real-time RT-PCR sensitivity was 2×101 copies. Using primers for the NCR region, 10 out of 16 (62.5 %) PBMCs were positive for negative-strand HCV RNA. Among the four samples collected from patients with SVR, negative-strand HCV RNA was found in two patient samples. Conclusions: Our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with SVR, in the presence of IL-2 and PHA, markedly enhanced the detection of HCV RNA replica-tive strands. Therefore, PBMCs may be reservoirs for recurrent hepatitis infection after SVR and antiviral treatment. However, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study.

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