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Title: Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

Abstract

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Authors:
;  [1]; ;  [1];  [1]; ;  [2]; ;  [3];  [4];  [5];  [1];  [1]
  1. Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan)
  2. Toray Industries, Inc., Kanagawa (Japan)
  3. Department of Pathology, National Institute of Infectious Diseases, Tokyo (Japan)
  4. Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medicine, Nagoya (Japan)
  5. Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba (Japan)
Publication Date:
OSTI Identifier:
22202600
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 395; Journal Issue: 4; Other Information: Copyright (c) 2010 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; CHROMATOGRAPHY; CULTURE MEDIA; ELECTRON MICROSCOPY; INFECTIOUS HEPATITIS; MICE; MORPHOGENESIS; MUTATIONS; PARTICLE STRUCTURE; PROTEINS; PURIFICATION; ULTRAFILTRATION; VACCINES; VIRUSES

Citation Formats

Takahashi, Hitoshi, Akazawa, Daisuke, Toray Industries, Inc., Kanagawa, Kato, Takanobu, Date, Tomoko, Shirakura, Masayuki, Toray Industries, Inc., Kanagawa, Nakamura, Noriko, Mochizuki, Hidenori, Tanaka-Kaneko, Keiko, Sata, Tetsutaro, Tanaka, Yasuhito, Mizokami, Masashi, Suzuki, Tetsuro, and Wakita, Takaji. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope. United States: N. p., 2010. Web. doi:10.1016/J.BBRC.2010.04.081.
Takahashi, Hitoshi, Akazawa, Daisuke, Toray Industries, Inc., Kanagawa, Kato, Takanobu, Date, Tomoko, Shirakura, Masayuki, Toray Industries, Inc., Kanagawa, Nakamura, Noriko, Mochizuki, Hidenori, Tanaka-Kaneko, Keiko, Sata, Tetsutaro, Tanaka, Yasuhito, Mizokami, Masashi, Suzuki, Tetsuro, & Wakita, Takaji. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope. United States. https://doi.org/10.1016/J.BBRC.2010.04.081
Takahashi, Hitoshi, Akazawa, Daisuke, Toray Industries, Inc., Kanagawa, Kato, Takanobu, Date, Tomoko, Shirakura, Masayuki, Toray Industries, Inc., Kanagawa, Nakamura, Noriko, Mochizuki, Hidenori, Tanaka-Kaneko, Keiko, Sata, Tetsutaro, Tanaka, Yasuhito, Mizokami, Masashi, Suzuki, Tetsuro, and Wakita, Takaji. 2010. "Biological properties of purified recombinant HCV particles with an epitope-tagged envelope". United States. https://doi.org/10.1016/J.BBRC.2010.04.081.
@article{osti_22202600,
title = {Biological properties of purified recombinant HCV particles with an epitope-tagged envelope},
author = {Takahashi, Hitoshi and Akazawa, Daisuke and Toray Industries, Inc., Kanagawa and Kato, Takanobu and Date, Tomoko and Shirakura, Masayuki and Toray Industries, Inc., Kanagawa and Nakamura, Noriko and Mochizuki, Hidenori and Tanaka-Kaneko, Keiko and Sata, Tetsutaro and Tanaka, Yasuhito and Mizokami, Masashi and Suzuki, Tetsuro and Wakita, Takaji},
abstractNote = {To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.},
doi = {10.1016/J.BBRC.2010.04.081},
url = {https://www.osti.gov/biblio/22202600}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 4,
volume = 395,
place = {United States},
year = {Fri May 14 00:00:00 EDT 2010},
month = {Fri May 14 00:00:00 EDT 2010}
}