scholarly journals Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1–3 Strains and Does Not Correlate with Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 389
Author(s):  
Andrea Galli ◽  
Santseharay Ramirez ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Liver Steatosis ◽  
Hcv Infection ◽  
Intracellular Lipid ◽  
Depth Analysis ◽  
Genotype 3A

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture.

scholarly journals Morphological changes in intracellular lipid droplets induced by different hepatitis C virus genotype core sequences and relationship with steatosis

Hepatology ◽  
2008 ◽  
Vol 48 (1) ◽  
pp. 16-27 ◽  
Author(s):  
Aurélie Piodi ◽  
Philippe Chouteau ◽  
Hervé Lerat ◽  
Christophe Hézode ◽  
Jean‐Michel Pawlotsky
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplets ◽  
Morphological Changes ◽  
Virus Genotype ◽  
Intracellular Lipid

scholarly journals Mutations Identified in the Hepatitis C Virus (HCV) Polymerase of Patients with Chronic HCV Treated with Ribavirin Cause Resistance and Affect Viral Replication Fidelity

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Niels Mejer ◽  
Ulrik Fahnøe ◽  
Andrea Galli ◽  
Santseharay Ramirez ◽  
Ola Weiland ◽  
...  
Keyword(s):  
Cell Culture ◽  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Prolonged Exposure ◽  
Resistance Mutations ◽  
Entry Site ◽  
Chronic Hcv ◽  
Genotype 3A

ABSTRACT Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.

scholarly journals Hepatitis C Virus Attenuates Mitochondrial Lipid β-Oxidation by Downregulating Mitochondrial Trifunctional-Protein Expression

2015 ◽  
Vol 89 (8) ◽  
pp. 4092-4101 ◽  
Author(s):  
Yutaka Amako ◽  
Tsubasa Munakata ◽  
Michinori Kohara ◽  
Aleem Siddiqui ◽  
Chris Peers ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Persistent Infection ◽  
Liver Steatosis ◽  
Hcv Infection ◽  
Host Cells ◽  
Type I ◽  
Mrna Levels ◽  
Mitochondrial Lipid ◽  
Mitochondrial Trifunctional Protein

ABSTRACTThe course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid β-oxidation. However, little is known about the effects of HCV on lipid β-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and β-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid β-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid β-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-of-function study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver.IMPORTANCEHCV infection triggers metabolic alterations, which lead to significant disease outcomes, such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid β-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid β-oxidation, host cells were less responsive to the ability of exogenously added IFN-α to suppress HCV replication. This suggests that interference with lipid β-oxidation may assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus-host interaction may lead to improvements in the current standard therapy.

scholarly journals microRNA-99a Restricts Replication of Hepatitis C Virus by Targeting mTOR and De Novo Lipogenesis

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 696 ◽  
Author(s):  
Eun Byul Lee ◽  
Pil Soo Sung ◽  
Jung-Hee Kim ◽  
Dong Jun Park ◽  
Wonhee Hur ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Accumulation ◽  
De Novo ◽  
Regulatory Element ◽  
Mammalian Target Of Rapamycin ◽  
De Novo Lipogenesis ◽  
Hcv Rna ◽  
Intracellular Lipid ◽  
Intracellular Lipid Accumulation

In this study, we investigated the role of microRNA-99a (miR-99a) in hepatitis C virus (HCV) replication and lipogenesis in hepatocytes. Cell-culture-derived HCV (HCVcc) infection caused down-regulation of miR-99a in Huh-7 cells, and the relative levels of miR-99a were significantly lower in the sera of the HCV-infected patients than in those of healthy controls. Transfection of miR-99a-5p mimics resulted in a decrease in the intracellular and secreted HCV RNA levels. It also caused a decreased mammalian target of rapamycin (mTOR) protein level and phosphorylation of its downstream targets in HCV-replicating cells. Sterol regulatory element binding protein (SREBP)-1c expression and intracellular lipid accumulation decreased when either miR-99a-5p mimics or si-mTOR was transfected in oleic acid-treated Huh-7 cells. Overexpression of mTOR rescued HCV RNA replication and lipid droplet accumulation in miR-99a-5p mimics-transfected HCV replicon cells. Our data demonstrated that miR-99a ameliorates intracellular lipid accumulation by regulating mTOR/SREBP-1c and causes inefficient replication and packaging of intracellular HCV.

scholarly journals Hepatitis C virus nonstructural protein 5A perturbs lipid metabolism by modulating AMPK/SREBP-1c signaling

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Ziyu Meng ◽  
Qiang Liu ◽  
Fujun Sun ◽  
Ling Qiao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Nonstructural Protein ◽  
Ectopic Expression ◽  
Serum Levels ◽  
Hepatic Lipid ◽  
Hepatic Lipid Accumulation ◽  
Nonstructural Protein 5A

Abstract Background Steatosis is an important clinical manifestation associated with chronic hepatitis C virus (HCV) infection. AMP-activated protein kinase (AMPK), a major mediator of lipid metabolism, regulates HCV-associated hepatic steatosis, but the underlying mechanisms remain obscure. Here we investigated the mechanism of HCV nonstructural protein 5A (NS5A)-induced lipid accumulation by the AMPK/SREBP-1c pathway. Methods We generated model mice by injecting recombinant lentiviral particles expressing the NS5A protein (genotype 3a) via the tail vein. The serum levels of alanine aminotransferase (ALT), free fatty acids (FFAs) and triglycerides (TG) were examined. H&E and Oil Red O staining were used to examine lipid droplets. Immunohistochemistry staining, quantitative real-time PCR and Western blotting were used to determine the expression of lipogenic genes. Results Our results showed that the serum levels of ALT, FFAs and TG, as well as the accumulation of hepatic lipid droplets, were increased significantly in mice infected with NS5A-expressing lentiviral particles. NS5A inhibited AMPK phosphorylation and increased the expression levels of sterol regulatory element binding protein-1c (SREBP-1c), acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) in vivo and in vitro. Further investigation revealed that pharmacological activation or ectopic expression of AMPK neutralized the upregulation of SREBP-1c, ACC1 and FASN, and ameliorated hepatic lipid accumulation induced by NS5A. Ectopic expression of SREBP-1c enhanced NS5A-induced hepatic lipid accumulation, which was dramatically reversed by pharmacological activation of AMPK. Conclusions Collectively, we demonstrate that NS5A induces hepatic lipid accumulation via the AMPK/SREBP-1c pathway.

scholarly journals FREQUENCY OF OCCURRENCE OF POLYMORPHIC VARIANTS OF IL28B GENE AND GENOTYPES OF HEPATITIS C VIRUS IN POPULATION OF YAKUTIA: CLINICAL OUTCOMES

2017 ◽  
pp. 86-92 ◽  
Author(s):  
S. I. Semenov ◽  
A. I. Fedorov ◽  
V. L. Osakovsky ◽  
S. S. Maksimova ◽  
F. A. Platonov
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Outcomes ◽  
Clinical Presentation ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Polymorphic Variants ◽  
Genotype 3A

Aim. Study clinical outcomes in patients with, chronic hepatitis C depending on genotype of hepatitis C virus (HCV) and IL28B gene polymorphism. Materials and methods. 592 individuals were examined, 75 of those had HCV RNAgenotypes determined by PCR. Genotyping of single nucleotide polymorphisms (SNP) - rs 12979860 (C/T) and rs8099917 (T/G) in IL28B gene was carried out by real-time PCR. Results. HCV RNA was detected in 72 examined residents of Yakutia. HCV lb genotype was determined in 74.2% of cases, 3a - in 11.4%, la and 2 - 5.7% each. Frequency of polymorph variant rsl2979860 CC was 72.2%, CT - 27.8%, rs8099917 TT - 61.1%, TG - 23.2%. Conclusion. Combination of HCV lb with polymorphic variants of IL28B gene rs.l.2979860 CC and rs8099917 CT showed a less aggressive course of the disease. On the other hand, HCV infection of individuals with genotype 3a and polymorphism rsl2979860 CC or rs809917 TT of IL28B gene showed a more severe clinical presentation. The presence of polymorph variants rs8099917 T/G and rs 12979860 C/T showed more severe clinical outcomes of HCV infection (viral load up to 19035212 copies, cirrhosis with ascite, hepatocarcinoma).

scholarly journals Persistent Transcriptional Alterations after Hepatitis C Virus Elimination in Cell Culture

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 79
Author(s):  
Victoria Castro ◽  
Gema Calvo ◽  
Juan Carlos Oliveros ◽  
Sofia Pérez-Del-Pulgar ◽  
Xavier Forns ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Host Cell ◽  
Hcv Infection ◽  
Virus Eradication ◽  
Transcriptional Profiles ◽  
Transcriptional Alterations ◽  
Infected Cells

Chronic hepatitis C virus (HCV) infection causes liver inflammation and fibrosis, which can lead to development of cirrhosis and hepatocellular carcinoma (HCC). The recent approval of direct-acting antiviral (DAA) drug combinations has revolutionized antiviral therapy against HCV. These drugs enable virus eradication in virtually all treated patients regardless of their genotype and liver disease status. Based on clinical parameters, it has been proposed that elimination of infected cells by reactivated immune responses may be dispensable for virus eradication in contrast to previously used interferon-based therapies. It is thus formally possible that the patients, who are declared cured, do indeed carry formerly infected cells that display irreversible alterations due to prolonged chronic HCV infection. Although transcriptional profiles of biopsies from cured patients have been previously studied, it is difficult to determine the precise mechanisms by which permanent alterations are established in the context of a heterogeneous tissue, often in patients with an underlying liver disease. Thus, we used cell culture models of persistent HCV infection to determine if HCV infection causes permanent transcriptional alterations in host cells after virus eradication. In these models, HCV infection causes profound alterations of host cell transcriptome that aim at regaining cellular homeostasis in the context of intracellular membrane rearrangements, interference with homeostatic processes, and persistent stress conditions, and permit cell survival even though the virus has colonized the host cell. In this context, we asked the question of whether the original homeostasis and original transcriptomic profile are regained in formerly infected cells after DAA treatment-mediated virus eradication. Our results indicate that a minor subset of transcriptional alterations persists even after virus eradication, suggesting that DAA-mediated eradication does not ensure the normalization of formerly infected cell homeostasis. Combined analysis of the transcriptional profiles in proliferating and growth-arrested cells suggests that several mechanisms underlie the establishment of permanent alterations.

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