Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins Vinca Icard1,2,3,4., Olivier Diaz1,2,3., Caroline Scholtes1,2,3,4, Laure Perrin-Cocon1,2,3, Christophe Ramie` re1,2,3,4, Ralf Bartenschlager5, Francois Penin6, Vincent Lotteau1,2,3,4, Patrice Andre´ 1,2,3,4* 1 Universite´ de Lyon, Lyon, France, 2 Inserm, U851, Lyon, France, 3 Universite´ de Lyon1, IFR128 BioSciences Lyon-Gerland, Lyon, France, 4 Hospices Civils de Lyon, Laboratoire de Virologie Nord, Lyon, France, 5 Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany, 6 CNRS, UMR 5086, Institut de Biologie et Chimie des Prote´ines, Lyon, France Abstract The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo- viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2- specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed. Citation: Icard V, Diaz O, Scholtes C, Perrin-Cocon L, Ramie`re C, et al. (2009) Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins. PLoS ONE 4(1): e4233. doi:10.1371/journal.pone.0004233 Editor: Brett Lindenbach, Yale University, United States of America Received July 3, 2008; Accepted December 3, 2008; Published January 21, 2009 Copyright: 2009 Icard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by a grant from the ANRS ‘‘Agence nationale de recherches sur le sida et les he´patites virales’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: patrice.andre@inserm.fr . These authors contributed equally to this work. Introduction Hepatitis C virus (HCV) infects an estimated 3% of the world population and frequently causes chronic infection often leading to cirrhosis and liver cancer. The virus was first isolated in 1989 by molecular biology techniques [1] and classified in the Hepacivirus genus within the Flaviviridae family, which includes the flaviviruses (e.g. yellow fever virus and Dengue virus), the pestiviruses (e.g., bovine viral diarrhoea virus), and GB viruses [2]. However, the high frequency of chronic infections and the very narrow host range limited to humans and chimpanzees sets HCV apart from the other flaviviruses. Since its discovery, many aspects of the HCV replication cycle as well as the pathophysiology of chronic hepatitis C have been described (for recent reviews, see [3,4]). Surprisingly, despite the possibility to propagate the virus in vitro, the precise nature of the HCV particle is still poorly understood in contrast to the flaviviruses that have been studied in great details [5–7]. Unique to HCV is the exceptional low density of the virus particles resulting from the association of the virus with lipoproteins [8,9]. Low density complexes of HCV and lipopro- teins are preferably observed in the blood of chronically infected patients while most of the viral particles produced in vitro have a density similar to that of flaviviruses [10,11]. The density of the blood circulating forms of HCV is very heterogeneous ranging from 1.25 to less than 1.06 g/mL. Particles with high density could correspond to naked capsids [12]. Particles in plasma density fraction around 1.15 g/mL may represent conventional viruses similar to those produced in Huh-7 cells that are derived from the highly replication competent JFH1 strain (HCVcc) [13–15]. Viral particles in density fractions below 1.06 g/mL are associated with apolipoprotein B (apoB) bearing triglyceride rich lipoproteins (TRL), namely the low, intermediate and very low density lipoproteins (LDL, IDL and VLDL, respectively) and chylomicrons [9,10,16–19]. This uncommon association of a virus with lipoproteins is of particular interest since viral particles of low density have a higher specific infectivity than PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4233