scholarly journals Probing the viral replication of HCV and XMRV : biochemical characterization, inhibition kinetics and role of host proteins in viral replication

2015 ◽  
Author(s):  
◽  
Tanyaradzwa P. Ndongwe
Keyword(s):  
Hepatitis C ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Rna Binding ◽  
Biochemical Characterization ◽  
Hcv Rna ◽  
Hepatitis C Infection ◽  
Human Hepatoma Cells ◽  
Murine Leukemia

The studies described in this thesis focus on RNA viruses XMRV and HCV. In Chapter II we focused on Xenotropic Murine leukemia-Related Virus (XMRV). We characterized the biochemical activity and kinetics of the XMRV reverse transcriptase and discovered key mechanistic differences between XMRV, Moloney murine leukemia virus (MoMLV), and human immunodeficiency virus (HIV-1) reverse transcriptase (RT) enzymes. In Chapter III and IV we focused on Hepatitis C Virus (HCV), the causative agent of hepatitis C infection. We studied the role of an antiviral host factor Mov10 in the viral replication of HCV. We demonstrate that Mov10 overexpression in human hepatoma cells restricts HCV RNA production in a fully infectious virus cell culture system, leading to decreased virus production over time. Additionally, overexpression of Mov10 in producer cells decreases the infectivity of the produced virus. Confocal microscopy shows HCV infection results in redistribution of endogenous Mov10 to circular structures surrounding lipid droplets, proximal to viral proteins core and NS5A. Finally, we demonstrate that the RNA-binding function of Mov10 is responsible for its antiviral effect. Decreasing Mov10 protein expression levels decreased HCV replication and infection levels. Our data reveal a complex balance between Mov10 and HCV. In Chapter IV we studied several aspects of HCV. (i) We discovered two novel small molecule inhibitors of the HCV helicase that have antiviral function. (ii) We discovered that Dcp2 is a novel HCV host restriction factor that can block HCV replication, and (iii) we provide insights into the mechanism(s) of action of approved and clinically advanced direct-acting antiviral agents (DAAs).

scholarly journals HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

2015 ◽  
Vol 90 (5) ◽  
pp. 2332-2344 ◽  
Author(s):  
Rong Yu ◽  
Darong Yang ◽  
Shaohua Lei ◽  
Xiaohong Wang ◽  
Xianghe Meng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Binding ◽  
High Mobility ◽  
Hcv Rna ◽  
Functional Domain ◽  
Hmgb1 Protein ◽  
Stem Loop

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

scholarly journals Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mun-Teng Wong ◽  
Steve S. Chen
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Ternary Complex ◽  
Choline Kinase ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Phosphatidylinositol 4

ABSTRACT In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication. IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

scholarly journals Genotype Dependence of Hepatitis C Virus Load Measurement in Commercially Available Quantitative Assays

1999 ◽  
Vol 37 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Janet Mellor ◽  
Anna Hawkins ◽  
Peter Simmonds
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Narrow Range ◽  
Antiviral Treatment ◽  
Hcv Rna ◽  
Clinical Specimens ◽  
Rna Transcripts ◽  
Relative Quantitation ◽  
Version 2.0

Standardization and genotype independence of methods used to quantify hepatitis C virus (HCV) RNA in clinical specimens are necessary for accurate assessment of the role of HCV quantitation as a prognostic marker for HCV infection and monitoring of the response to antiviral treatment. Commercially available methods used to measure HCV loads include PCR-based (Roche Monitor) and hybridization-based (Quantiplex bDNA-2) methods. Recently, a new version of the Roche Monitor assay (version 2.0) has become available; it has been modified to achieve more equal quantitation of different HCV genotypes. Consistent with previous reports, Roche Monitor version 1.0 substantially underestimated concentrations of RNA transcripts of types 2b, 3a, 4a, 5a, and 6a and virus loads in individuals infected with genotypes 2 to 6 relative to reference tests. However, version 2.0 achieved equivalent quantitation of each genotype over a narrow quantitative range (103 to 5 × 105 copies of RNA/ml) but significantly underestimated RNA concentrations above this range. The assay showed an equivalent inability to quantify high levels of HCV RNA in plasma samples, and this was responsible for the falsely narrow range of virus loads detected in HCV-infected individuals. In contrast, the Chiron bDNA-2 assay could only measure RNA concentrations in the upper quantitative range (2 × 105 to 5 × 107 copies of RNA/ml) but showed equivalent sensitivity for genotypes 1 to 5; however, concentrations of type 6a RNA transcripts and virus loads in clinical specimens from individuals infected with type 6a were underestimated by a factor of 2 to 4. Differences were observed between PCR- and hybridization-based assays in their relative quantitation of HCV RNA transcripts and HCV genomic RNA, which may cause problems with the use of transcripts for interassay calibration.

scholarly journals Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

2012 ◽  
Vol 93 (2) ◽  
pp. 247-258 ◽  
Author(s):  
Anna Pasetto ◽  
Lars Frelin ◽  
Anette Brass ◽  
Anila Yasmeen ◽  
Sarene Koh ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Cytotoxic T Lymphocyte ◽  
Hcv Rna ◽  
High Avidity ◽  
Immune Recognition ◽  
Structural Protein ◽  
Human Hepatoma Cells

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

scholarly journals Spontaneous Clearance of Chronic HCV: The Key Ending Left in the Dark

2019 ◽  
Vol 7 (10) ◽  
pp. 1657-1659
Author(s):  
Nikola Hristov Mumdzhiev ◽  
Daniela Valerieva Radicheva ◽  
Mariana Penkova Radicheva ◽  
Rumen Valchev Tenev ◽  
Zlatina Dimitrova Vasileva
Keyword(s):  
Hepatitis C ◽  
Hcv Rna ◽  
Spontaneous Clearance ◽  
Hepatitis C Infection ◽  
Positive Serology ◽  
Mild Disease ◽  
Spontaneous Hcv Clearance ◽  
Chronic Hcv ◽  
Direct Acting ◽  
Special Circumstances

BACKGROUND: Hepatitis C is the second leading cause of liver cirrhosis and hepatocellular carcinoma. Although the discovery of direct-acting agents made the disease curable, HCV elimination can be achieved solely by the host’s immunologic arsenal. CASE REPORT: We report the case of a 29-year-old woman with chronic hepatitis C infection - elevated transaminases, positive serology. HCV was detectable on two occasions, and histology showed mild disease - A1F1. Upon follow up and without any treatment, the patient achieved spontaneous clearance confirmed by two consecutive undetectable HCV RNA tests. Spontaneous HCV clearance rarely occurs – 0.5% per person-year. This is sometimes accompanied by special circumstances like additional disease or medical interventions. Host factors like gender and interleukin-28B polymorphisms have been known to contribute to clearance. Viral factors like HCV RNA levels are also a factor. The characteristics of host-viral interplay – age of acquisition and fibrosis stage – cannot be overlooked. CONCLUSION: All of the abovementioned factors contribute to the complex immunological interaction between virus and host and the result, although rarely can be spontaneous clearance.

scholarly journals Increased levels of circulating IL-10 in persons recovered from hepatitis C virus (HCV) infection compared with persons with active HCV infection

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Dorcas Ohui Owusu ◽  
Richard Phillips ◽  
Michael Owusu ◽  
Fred Stephen Sarfo ◽  
Margaret Frempong
Keyword(s):  
Hepatitis C ◽  
Spontaneous Recovery ◽  
Predictive Marker ◽  
Serum Levels ◽  
Hcv Infection ◽  
Tnf Alpha ◽  
Hepatitis C Infection ◽  
Ifn Gamma ◽  
The Relationship

Abstract Objective Approximately 70% of all hepatitis C (HCV) infections develop chronic disease. Active or exacerbated chronic hepatitis C infection subsequently progress to liver disease. The role of T-cells secretions in achieving viral clearance is still not well understood. Thus, the current study was set to determine the relationship between the T cell cytokine profiles, biochemical parameters and persistent HCV infection or spontaneous recovery. Results Twenty-five percent (41/163) of the anti-HCV positive participants had recovered from HCV and had significantly higher concentration of IL-10 compared to those with active HCV infection (P < 0.012). Other circulating cytokines measured; IL-2, IFN gamma, TNF alpha, IL-5 and IL-17 were similar in both groups. Participants with active HCV infection had significantly higher aspartate transaminase (AST) (35 units) and alanine transaminase (46 units) compared to those in the recovered state (P < 0.001). Thus, serum levels of IL10 could be explored in larger prospective cohort study as a predictive marker of recovering from an active HCV infection.

scholarly journals TNRC6 proteins modulate hepatitis C virus replication by spatially regulating the binding of miR-122/Ago2 complexes to viral RNA

2019 ◽  
Vol 47 (12) ◽  
pp. 6411-6424 ◽  
Author(s):  
You Li ◽  
Li Wang ◽  
Efraín E Rivera-Serrano ◽  
Xian Chen ◽  
Stanley M Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Spatial Organization ◽  
Host Factor ◽  
Viral Rna ◽  
Fine Tuning ◽  
Hcv Rna ◽  
Human Hepatoma Cells ◽  
Translational Activity ◽  
The Stability

AbstractThe liver-specific microRNA, miR-122, is an essential host factor for replication of the hepatitis C virus (HCV). miR-122 stabilizes the positive-strand HCV RNA genome and promotes its synthesis by binding two sites (S1 and S2) near its 5′ end in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using an unbiased quantitative proteomics screen, we identified the TNRC6 protein paralogs, TNRC6B and TNRC6C, as functionally important but redundant components of the miR-122/Ago2 host factor complex. Doubly depleting TNRC6B and TNRC6C proteins reduced HCV replication in human hepatoma cells, dampening miR-122 stimulation of viral RNA synthesis without reducing the stability or translational activity of the viral RNA. TNRC6B/C were required for optimal miR-122 host factor activity only when S1 was able to bind miR-122, and restricted replication when S1 was mutated and only S2 bound by miR-122. TNRC6B/C preferentially associated with S1, and TNRC6B/C depletion enhanced Ago2 association at S2. Collectively, these data suggest a model in which TNRC6B/C regulate the assembly of miR-122/Ago complexes on HCV RNA, preferentially directing miR-122/Ago2 to S1 while restricting its association with S2, thereby fine-tuning the spatial organization of miR-122/Ago2 complexes on the viral genome.

scholarly journals Risk Factors Contributing to the Occurrence and Recurrence of Hepatocellular Carcinoma in Hepatitis C Virus Patients Treated with Direct-Acting Antivirals

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 175
Author(s):  
Sara Kishta ◽  
Ashraf Tabll ◽  
Tea Omanovic Kolaric ◽  
Robert Smolic ◽  
Martina Smolic
Keyword(s):  
Risk Factors ◽  
Hepatocellular Carcinoma ◽  
Hepatitis C ◽  
Dna Ploidy ◽  
Hcv Rna ◽  
Direct Acting Antivirals ◽  
Hepatic Cells ◽  
Chronic Hcv ◽  
Direct Acting

Although hepatitis C virus (HCV) RNA may be eliminated from blood circulation by direct-acting antivirals (DAA) therapy as assessed by real-time polymerase chain reaction (PCR), HCV RNA can still be present in liver tissue, and this is known as occult HCV. There has been a lot of controversy surrounding the recurrence of hepatocellular carcinoma (HCC) after DAA treatment of hepatic cells infected with chronic HCV. One of the main risk factors that leads to de novo HCC is the chronicity of HCV in hepatic cells. There are many studies regarding the progression of HCV-infected hepatic cells to HCC. However, there is a lack of research on the different molecular mechanisms that lead to the progression of chronic HCV infection to HCC, as well as on the effect of HCV on the alteration of DNA ploidy, which eventually leads to a recurrence of HCC after DAA treatment. In this review article, we will address some risk factors that could lead to the development/recurrence of HCC after treatment of HCV with DAA therapy, such as the role of liver cirrhosis, the alteration of DNA ploidy, the reactivation of hepatitis B virus (HBV), the role of cytokines and the alteration of the immune system, concomitant non- alcoholic fatty liver disease (NAFLD), obesity, alcohol consumption and also occult HCV infection/co-infection. Clinicians should be cautious considering that full eradication of hepatocarcinogenesis cannot be successfully accomplished by anti-HCV treatment alone.

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