scholarly journals Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

2021 ◽  
Vol 21 (6) ◽  
Author(s):  
Shaomei Zhu ◽  
Bochao Liu ◽  
Yuxia Xu ◽  
Ling Zhang ◽  
Zhengyuan Xia ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Western Blot ◽  
Affinity Chromatography ◽  
Magnetic Separation ◽  
Immune Electron Microscopy ◽  
Serum Free ◽  
Purification Methods ◽  
Heparin Affinity

Background: The traditional ultracentrifugation purification method of hepatitis C virus (HCV) particles requires special equipment, limiting its wide application. Therefore, more effective and convenient methods for HCV are needed. Objectives: The present study aimed to establish simple and effective purification methods for HCV. Methods: The infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcription-quantitative polymerase chain reaction and immunofluorescence. After concentration by centrifugal filter devices, HCV particles were purified by heparin-affinity chromatography and magnetic separation technique. The purified viruses were detected by the western blot and immune-electron microscopy. Results: The infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5 × 104 FFU/mL, and HCV ribonucleic acid load was 3.946 × 106 IU/mL in 30 days of cell culture post-transfection. After purification by heparin-affinity chromatography or magnetic separation method, viral particles were visualized with spherical morphology and an average diameter of 55 nm assessed by electron microscopy. The viruses were confirmed by the western blot and immune-electron microscopy with specific antibodies to HCV. Conclusions: The heparin-affinity chromatography and magnetic separation methods were established for the purification of HCV, which were simple and efficient methods for the stable purification of HCV particles on a large scale.

Serum-free cell culture system supplemented with lipid-rich albumin for hepatitis C virus (strain O of genotype 1b) replication

2007 ◽  
Vol 125 (2) ◽  
pp. 162-168 ◽  
Author(s):  
Ken-ichi Abe ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Hiromichi Dansako ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Strain ◽  
Culture System ◽  
Free Cell ◽  
Cell Culture System ◽  
Serum Free ◽  
Genotype 1B

scholarly journals Expression of Hepatitis C Virus E2 Ectodomain in E. coli and Its Application in the Detection of Anti-E2 Antibodies in Human Sera

2004 ◽  
Vol 36 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Jing Liu ◽  
Xin-Xin Zhang ◽  
Shen-Ying Zhang ◽  
Min Lu ◽  
Yu-Ying Kong ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Western Blot ◽  
Mammalian Cells ◽  
Rabbit Antiserum ◽  
Hcv Rna ◽  
Target Cells ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Human Sera

Abstract The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385–730) with a four-amino-acid mutation (aa 568–571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2-specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glycoproteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385–565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.

scholarly journals Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

2010 ◽  
Vol 91 (8) ◽  
pp. 1919-1930 ◽  
Author(s):  
Pierre Bonnafous ◽  
Marie Perrault ◽  
Olivier Le Bihan ◽  
Birke Bartosch ◽  
Dimitri Lavillette ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Fusion ◽  
Magnetic Beads ◽  
Neutral Ph ◽  
Transmission Electron ◽  
Magnetic Nanobeads

Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1–E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1–E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1–E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.

Immune complex of hepatitis C virus particles detected by immunogold electron microscopy

2006 ◽  
Vol 41 (8) ◽  
pp. 807-808
Author(s):  
Masahiko Kaito ◽  
Esteban C Gabazza ◽  
Naoki Fujita ◽  
Hideaki Tanaka ◽  
Shozo Watanabe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Complex ◽  
Immunogold Electron Microscopy ◽  
Virus Particles

scholarly journals A Single-Amino-Acid Mutation in Hepatitis C Virus NS5A Disrupting FKBP8 Interaction Impairs Viral Replication

2008 ◽  
Vol 82 (7) ◽  
pp. 3480-3489 ◽  
Author(s):  
Toru Okamoto ◽  
Hiroko Omori ◽  
Yuuki Kaname ◽  
Takayuki Abe ◽  
Yorihiro Nishimura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Fluorescence Microscopy ◽  
Viral Replication ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Single Amino Acid ◽  
Single Amino Acid Residue

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val121 to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala121 with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.

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