Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Jul 8, 2016; 8(19): 796-814
Published online Jul 8, 2016. doi: 10.4254/wjh.v8.i19.796
Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway
Miriam Triyatni, Edward A Berger, Bertrand Saunier
Miriam Triyatni, Roche Innovation Center, Roche Pharma Research and Early Development, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
Miriam Triyatni, Edward A Berger, Bertrand Saunier, Molecular Structure Section, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD 20892-3210, United States
Bertrand Saunier, Unité de Virologie Structurale, Institut Pasteur and CNRS UMR 3569, 75724 Paris, France
Author contributions: Triyatni M designed and performed experiments and analyzed the data; Berger EA provided facilities and resources for research, analyzed the data, and contributed to editing the manuscript; Saunier B designed most and performed some experiments, analyzed the data and wrote the manuscript.
Supported by Intramural Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases (Project No. 1 ZIA AI000733-15: Enveloped Virus Glycoprotein/Receptor Interactions) to Edward A Berger, PhD (MSS, LVD, DIR, NIAID); and ORISE Senior Fellow award (Award No. 1238-1238-03: Department of Energy/Oak Ridge Institute for Science and Education) to Bertrand Saunier, MD, PhD.
Institutional review board statement: Approved the use of serum IgG of a patient cured from an HCV infection for in vitro studies (Hôpital and Institut Cochin, Paris).
Institutional animal care and use committee statement: Not applicable.
Conflict-of-interest statement: Saunier B, Triyatni M and Berger EA are co-inventors on NIH-owned patent #US 9052321 B2 on the HCV particle production system.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Bertrand Saunier, MD, PhD, Unité de Virologie Structurale, Institut Pasteur and CNRS UMR 3569, Centre François Jacob, 28 rue du Docteur Roux, 75724 Paris, France. bsaunier@pasteur.fr
Telephone: +33-01-45688855 Fax: +33-01-45688993
Received: January 31, 2016
Peer-review started: February 1, 2016
First decision: March 25, 2016
Revised: May 16, 2016
Accepted: June 1, 2016
Article in press: June 3, 2016
Published online: July 8, 2016
Processing time: 156 Days and 11.9 Hours
Abstract

AIM: To determine if calnexin (CANX), RAB1 and alpha-tubulin were involved in the production of hepatitis C virus (HCV) particles by baby hamster kidney-West Nile virus (BHK-WNV) cells.

METHODS: Using a siRNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observed in thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.

RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.

CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.

Keywords: Membrane rearrangements; Hepatitis C virus; Flavivirus replicon; Virus assembly and secretion; Host cellular factors

Core tip: Our system for production of authentic infectious hepatitis C virus (HCV) in non-humanized, non-hepatic cells involves the rearrangement of inner cellular membranes triggered by the replication of flaviviruses. The present results suggest that this feature relies on the re-wiring of host factors that usually contribute to the secretion of glycoproteins to generate an unusual secretory pathway. This model offers a new way to study the properties of free HCV particles, i.e., independently from lipoproteins.