Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

Abstract

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was Nterminally tagged with a FLAG epitope. This virus, designated Jc1E2 FLAG , produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2 FLAG particles to resemble the one very low-and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2 FLAG particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

Hepatitis c virus and host cell lipids: An intimate connection

“…167 This result is in keeping with the notion that assembly and release of HCV particles are tightly linked to host cell lipoproteins and lipids. 162,[168][169][170][171][172] Moreover, the nature of the lipidic constituents of HCV particles appears to depend on the host cell in which the virus is produced.…”

Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

“…The presence of apoB in viral particles issued from Huh-7.5 cells is still a matter of debate. Although some studies have indicated that apoB is dispensable for production of infectious particles (52) and is barely detected in viral particles derived from Huh-7.5 (9,53), other studies have revealed its importance in HCV assembly and maturation (1,11,54). Moreover, the presence of apoB on secreted HCV particles has been demonstrated in viruses derived from cultured primary hepatocytes (32) or from long term cultures of Huh-7.5 cells differentiated with human serum-containing culture media (55) and in viruses recovered from patients (7,8).…”

“…To separate viral particles from lipoproteins, we performed co-IP assays using antibodies against apolipoproteins apoE and apoB, and we quantified the captured particles by HCV RNA-specific RT-quantitative PCR. We decided to use this approach because previous papers showed that the initial capture of viral particles with anti-E2 antibodies was inefficient probably because of poor epitope accessibility (9,10). We first performed co-IP assays on non-fractionated samples using apoE or apoB antibodies.…”

“…However, most of these cell lines have impaired production of VLDL because they express pre-VLDLs that are not fully lipidated and that do not fuse with apoE-containing luminal lipid droplets (30). The resulting HCVcc particles are thus poorly associated with apoB (9), and their buoyant density profile is significantly different compared with in vivo recovered viruses (31,32). As lipid and apolipoprotein components are key factors influencing HCV biology, this highlights the need to better understand how they are distributed within the different forms of HCV particles circulating in vivo and how such distributions functionally influence their entry properties.…”

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

“…[12][13][14] Second, the phospholipid compositions of HCVcc and TRL share similar characteristics, whereas they strikingly differ from those of cellular membranes or envelopes of virus that assemble at cellular membranes. [15][16][17] Furthermore, lipoprotein lipases that specifically hydrolyse lipoprotein triacylglycerol modify HCVcc biochemical and physical features and decrease their infectivity. 18,19 Thus, both in vivo-produced and in vitro-produced HCV particles share many characteristics of lipoprotein association, but with differences in the extent of apoB association.…”