scholarly journals mTORC1 Restricts Hepatitis C Virus Replication Through ULK1-mediated Suppression of miR-122 and Facilitates Post-replication Events

2018 ◽  
Author(s):  
Manish Kumar Johri ◽  
Hiren Vasantrai Lashkari ◽  
Dhiviya Vedagiri ◽  
Divya Gupta ◽  
Krishnan Harinivas Harshan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infection ◽  
Virus Replication ◽  
Viral Rna ◽  
Hcv Rna ◽  
Significant Drop ◽  
Mechanistic Target Of Rapamycin ◽  
Mtorc1 Activation ◽  
Rna Levels

ABSTRACTMechanistic target of rapamycin (mTOR) is an important kinase that assimilates several upstream signals including viral infection and facilitates appropriate response by the cell through two unique complexes mTORC1 and mTORC2. Here, we demonstrate that mTORC1 is activated early during HCV infection as antiviral response. Pharmacological inhibition of mTORC1 promoted HCV replication as suggested by elevated levels of HCV (+) and (-) RNA strands. This was accompanied by significant drop in extracellular HCV RNA levels indicating defective post-replication stages. The increase in viral RNA levels failed to augment intracellular infectious virion levels, suggesting that mTORC1 inhibition is detrimental to post-replication steps. Lower infectivity of the supernatant confirmed this observation. Depletion of Raptor and ULK1 accurately reproduced these results suggesting that mTORC1 imparted these effects on HCV through mTORC1-ULK1 arm. Interestingly, ULK1 depletion resulted in increased levels of miR-122, a critical host factor for HCV replication, thus revealing a new mechanism of regulation by ULK1. The binary effect of mTORC1 on HCV replication and egress suggests that mTORC1-ULK1 could be critical in replication: egress balance. Interestingly we discover that ULK1 depletion did not interfere with autophagy in Huh7.5 cells and hence the effects on HCV replication and post-replication events are not resultant of involvement of autophagy. Our studies demonstrate an overall ULK1 mediated anti-HCV function of mTORC1 and identifies an ULK1-independent autophagy that allows HCV replication in spite of mTORC1 activation.

scholarly journals Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon

2009 ◽  
Vol 83 (13) ◽  
pp. 6383-6390 ◽  
Author(s):  
Harel Dahari ◽  
Bruno Sainz ◽  
Alan S. Perelson ◽  
Susan L. Uprichard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Second Phase ◽  
Inhibition Kinetics ◽  
Rna Levels

ABSTRACT Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-α), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-α concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-α treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-α treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began ∼12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-α-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-α inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-α. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-α-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-α suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-α inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-α may be inhibiting HCV in vivo.

Serum Hepatitis C Virus RNA Levels and Histologic Findings in Liver Allografts With Early Recurrent Hepatitis C

2000 ◽  
Vol 124 (11) ◽  
pp. 1623-1627 ◽  
Author(s):  
Young Nyun Park ◽  
Peter Boros ◽  
David Y. Zhang ◽  
Patricia Sheiner ◽  
Leona Kim-Schluger ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Biopsy ◽  
Activity Index ◽  
Hcv Rna ◽  
Recurrent Hepatitis ◽  
Biopsy Specimens ◽  
Histologic Activity Index ◽  
Recurrent Hepatitis C ◽  
Rna Levels

Abstract Background.—Histopathologic features of early recurrent hepatitis C after orthotopic liver transplantation (OLTx) may be modified by immunosuppressive therapy or complicated by other conditions. Hepatitis C virus (HCV) RNA level usually increases after OLTx, but its correlation to histologic findings is not clear. Objective.—To evaluate the histologic findings of early recurrent hepatitis C in liver allografts and its correlation to serum HCV RNA level. Methods.—We studied 14 patients who underwent OLTx for chronic HCV infection. Thirty liver biopsy specimens and HCV RNA levels of 22 corresponding plasma samples obtained during the first 6 months following OLTx were analyzed. The control group (9 patients, 25 biopsy specimens) was chosen at random from patients with chronic liver disease other than HCV who were undergoing OLTx, and all tested negative for HCV RNA by polymerase chain reaction after OLTx. Results.—Statistically significant pathological features of early recurrent HCV infection were the number of acidophilic bodies, piecemeal necrosis, lymphocyte predominance in the portal tracts, and fibrous septum. These findings and histologic activity index scores increased with time after OLTx. The HCV RNA levels determined by branched DNA assay showed no significant correlation with histologic features. However, patients with higher histologic activity index scores tended to have higher RNA levels. Conclusions.—Liver biopsy specimens are helpful for the diagnosis or confirmation of early recurrent hepatitis C in liver allografts, but serial biopsy specimens are sometimes required for definite diagnosis. The HCV RNA levels are usually higher in patients who display signs of more severe liver damage.

scholarly journals Characterization of Occult Hepatitis C Virus (Hcv) Infection among Hemodialysis Patient

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Siti Nurul Fazlin Abdul Rahman ◽  
Hairul Aini binti Hamzah ◽  
Mohammed Imad Mustafa ◽  
Mohamed Hadzri Hasmoni
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hemodialysis Patients ◽  
Hcv Infection ◽  
Viral Rna ◽  
Cell Counting ◽  
Hcv Rna ◽  
Viral Rnas ◽  
Occult Hepatitis ◽  
Occult Hepatitis C

Introduction: The existence of new entity called occult hepatitis C virus (HCV) has become a raising and escalating concern among healthcare professionals worldwide. It is defined by the presence of viral RNA in liver and/or peripheral blood mononuclear cells (PBMCs) within non HCV-infected patients. Previous study had shown the occult HCV is infectious and capable of transmitting the virus to another host. Till today, HCV infection remains common among hemodialysis patients despite having the best preventive plans. Because of this, there is a significant concern about the source of viral transmission. The aim of the study was to identify and characterize occult HCV infection in PBMC sample of hemodialysis patients. This was an observational and cross sectional study. Materials and method: PBMCs were isolated from the whole blood using Ficoll-gradient centrifugation technique. The PBMCs were then subjected for cell counting and stored in -70O C until further used. HCV RNA were extracted from these cells and viral RNA were subjected for molecular assays, immune cells analysis and cells culture. Results: PBMCs were isolated from eleven (11) study patients and five (5) anti-HCV positive (control) patients. By using automated flow cytometry, PBMCs of each sample were counted and the average number of cells obtained range from 2x104 to 5x106 cells/ ml. Viral RNAs were extracted and quantitatively measured by using NanoDrop Spectrophotometers. The viral RNAs concentration obtained were between 24.7 and 258.9 ng/ml. The RNAs would be subjected for purification (ethanol precipitation) and further assays. Conclusion: The final findings might contribute to the clinical management of dialysis patients.

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 Poly(I:C) and Lipopolysaccharide Innate Sensing Functions of Circulating Human Myeloid Dendritic Cells Are Affected In Vivo in Hepatitis C Virus-Infected Patients

2007 ◽  
Vol 81 (11) ◽  
pp. 5537-5546 ◽  
Author(s):  
Ian Gaël Rodrigue-Gervais ◽  
Loubna Jouan ◽  
Geneviève Beaulé ◽  
Dominike Sauvé ◽  
Julie Bruneau ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Poly I:C ◽  
Hcv Rna ◽  
Genomic Rna ◽  
Tnf Α ◽  
Wide Range ◽  
Rna Levels

ABSTRACT The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log10, 5.0 per 106 cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-α− IL-12− cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs (∼6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.

scholarly journals MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Isaac Ruiz ◽  
Quentin Nevers ◽  
Eva Hernández ◽  
Nazim Ahnou ◽  
Rozenn Brillet ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatoma Cell ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Cysteinyl Leukotriene ◽  
Leukotriene Receptor ◽  
Cysteinyl Leukotriene Receptor ◽  
Dose Dependent ◽  
Rna Levels

ABSTRACT The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10. MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

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