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1 IFR 128 Biosciences Lyon Gerland, INSERM U503, 21 avenue Tony Garnier, 69007 Lyon, France
2 Université Pierre et Marie Curie, INSERM, UMRS 505, Paris, France
3 Service d'Hépato-Gastro-Entérologie, Hôtel Dieu, Hospices Civils de Lyon, France
4 Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, France
Correspondence
Patrice André
andre{at}cervi-lyon.inserm.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, in contrast to flaviviruses and closely related viruses, cell culture of HCV remained problematic for 15 years and this lack of an appropriate in vitro replication system and of a small-animal model impeded the understanding of HCV structure and replication cycle. Therefore, most of our knowledge of the virus cell receptors and of HCV RNA replication relied on pseudotyped viruses and on biscistronic and subgenomic replicons, which do not allow the study of HCV assembly or secretion, or the identification of the elusive nature of the virion. Recently, complete replication and production of infectious HCV particles in tissue culture were performed with HCV genotype 2a full-length replicons derived from a patient with fulminant hepatitis (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005). This major breakthrough identified a viral structure with size, morphology and density (1.15 g ml–1) appropriate for a member of the family Flaviviridae. The structure of these virions probably matches that of virions found in the plasma of chronically infected patients, with a density of 1.15 g ml–1 and recognized by anti-HCV envelope antibodies (Kaito et al., 1994; Petit et al., 2005; Takahashi et al., 1992).
Several forms of HCV particles coexist in the plasma of infected patients (Carrick et al., 1992; Kanto et al., 1994; Miyamoto et al., 1992) with a wide range of density (from 1.30 g ml–1 to an unusual low density of <1.06 g ml–1). Low-density viral particles are of particular interest, as they correlate with plasma infectivity in chimpanzees (Bradley et al., 1991; Hijikata et al., 1993). Interestingly, chimpanzee infection with in vitro-produced HCV with a density of 1.14 g ml–1 led to plasma HCV particles whose specific infectivity was recovered in fractions of lower density, indicating that a shift to lower buoyant density was correlated with an increased specific infectivity of HCV grown in vitro (Lindenbach et al., 2006). The low density of some HCV particles was attributed to an association of the virus with triacylglycerol (TG)-rich lipoproteins (TRLs) (Prince et al., 1996; Thomssen et al., 1992). Proportions of plasma HCV RNA found associated with TRLs vary from patient to patient, with a mean value close to 40 %, but can reach almost 100 % for some patients (André et al., 2002; Nielsen et al., 2004, 2006; Thomssen et al., 1992, 1993). Some of these TRL-like structures have been described as lipo-viroparticles (LVPs), whose structure and origin remain to be better defined (André et al., 2002; Nielsen et al., 2006).
TRLs are very-low-density particles (d
1.006 g ml–1) made of a hydrophobic core of neutral lipids, TG and cholesterol esters, surrounded by a monolayer of phospholipids (PL) and free cholesterol, associated with apoB and other apolipoproteins (Fisher & Ginsberg, 2002). TRLs are formed by the assembly of one molecule of apoB with TG within the endoplasmic reticulum lumen. apoB is a non-exchangeable apolipoprotein that remains associated with the particle until its capture and internalization by lipoprotein receptors. In humans, hepatocytes secrete very-low-density lipoproteins (VLDLs), which comprise one apoB100 molecule per particle, whereas enterocytes secrete another class of TRL, chylomicrons, which contain one molecule of apoB48, the truncated form of apoB resulting from the enterocyte-specific editing of apoB mRNA (Patterson et al., 2003). In the circulation, TRLs are subjected to TG hydrolysis by lipoprotein lipase, releasing free fatty acids, the remodelling of surface lipids and of exchangeable apolipoproteins A, C and E. These modifications give rise to particles of smaller size and higher density, i.e. remnants from chylomicrons and intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) from VLDLs.
LVPs are low-density, globular, HCV RNA-containing particles covered with natural antibodies allowing their purification from plasma low-density fractions (d<1.055 g ml–1) (André et al., 2002). They are rich in TG and contain internal structures that appear as capsids recognized by anti-core protein antibodies after delipidation. Binding and entry of purified LVPs into cells was competed by native VLDLs and by anti-apoB and anti-apoE antibodies, and increased by upregulation of the LDL receptor (Agnello et al., 1999; André et al., 2002). Therefore, LVPs appear to display some features of TRL-like structures. To further characterize the TRL-like nature of LVPs, the apolipoprotein composition of LVPs was analysed, as well as their lipid composition during the dynamic transition from the pre-prandial to the post-prandial period.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Blood samples and patients.
Plasma samples from HCV-negative and HCV-positive blood donors were obtained from the Etablissement de Transfusion Sanguine, Lyon, France. Eight volunteers attending the Service d'Hépato-Gastro-Entérologie at the Hôtel Dieu Hospital, Lyon, France, were selected in accordance with hospital ethics committee statements and enrolled in the study of the transition from the pre- to post-prandial states and of the lipidomic analysis of their plasma viral population (Table 1). These patients were chronically HCV-infected and had not been given antiviral therapy for at least 6 months. HCV genotypes were determined by sequencing of the 5'-untranslated region, and presence of cryoglobulinaemia was checked by routine laboratory examination. Patients were given a breakfast of a 900 kcal meal containing 30 % fat after an overnight fasting. Peripheral blood was drawn just before breakfast and 90 min after the first phlebotomy. EDTA was added to 0.1 mM final concentration and samples were processed immediately.
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LVP purification.
LVP purification was performed as described previously (André et al., 2002). Briefly, protein A-coated magnetic beads (Miltenyi Biotec) were incubated at room temperature with 2 ml low-density fractions in PBS with gentle rocking for 30 min. Samples were then passed through a magnetic column (Miltenyi Biotec), washed with PBS and collected in 500 µl Dulbecco's modified Eagle's medium/0.2 % BSA. Immunocaptured particles (purified LVPs) were stored at 4 °C in the dark in the presence of 2 % inhibitor cocktail.
Protein, apoB and lipid quantification.
Protein concentration was determined according to the Lowry method and calculated from a calibration curve by using BSA as a standard. apoB concentration in low-density fractions and sera was determined by using immunochemical kits (ApoB kit, bioMérieux, or ApoB kit, SFRI Diagnostics). Total cholesterol, PL and TG concentrations in sera were calculated with Cholesterol RTU, Phospholipides Enzymatique PAP 150 and Triacylglycerols Enzymic PAP 150 kits (bioMérieux), with the inclusion of standard curves to calculate the concentrations.
apoB concentrations in purified LVPs were determined by ELISA. Ninety-six-well flat-bottomed ELISA plates (Maxisorb; Nunc) were coated overnight at 4 °C with 100 µl monoclonal anti-human apoB antibody (5 µg ml–1; clone 1609) in PBS and then saturated with 2 % BSA for 1 h. Samples were first incubated for 30 min at room temperature in PBS/0.2 % BSA supplemented with 10 µg human IgG ml–1 before being distributed at 100 µl per well. After 2 h incubation at 37 °C and washing with PBS/0.05 % Tween 20, peroxidase-conjugated goat anti-human apoB antibody (1.6 µg ml–1) 100 µl per well in PBS/0.2 % BSA was added for 90 min at 37 °C. The plates were washed and o-phenylenediamine substrate was added (150 µl per well). The reaction was revealed for 10 min and A490 was read. Standard curves were established with LDL dilutions ranging from 2 to 100 ng apoB ml–1. Controls included human IgG-saturated protein A-coated magnetic beads prepared under the same conditions.
PL and TG compositions of LVP and low-density fractions were determined by gas chromatography quantification of their fatty acid content. Diheptadecanoyl phosphatidylcholine and triheptadecanoyl glycerol were added to LVPs and low-density fractions before lipid extraction as internal standards. Lipid extracts obtained from 200 µl LVPs or 100 µl low-density fraction were separated on Silica Gel G60 plates (Merck) with the solvent system hexane/diethyl ether/acetic acid (60 : 40 : 1, by vol.). The silica-gel areas corresponding to PL and TG were scraped off and transmethylated. Briefly, 1 vol. 5 % H2SO4 in methanol was added to the scraped silica gel and transmethylation was carried out at 100 °C for 90 min in screw-capped tubes. The reaction was terminated by the addition of 1.5 vols ice-cold 5 % (w/v) K2CO3 and the fatty acid methyl esters were extracted with isooctane and analysed by using a PerkinElmer Life Sciences chromatograph model 5830, equipped with a capillary column (30 mx0.32 mm; Supelco) and flame-ionization detection. The column was two-step-programmed from 135 to 160 °C at 2 °C min–1 and from 160 to 205 °C at 1.5 °C min–1; the detection temperature was maintained at 250 °C. The vector gas was helium at a pressure of 0.8 p.s.i. (5520 Pa). Peaks were identified by using standard fatty acid methyl esters and the absolute amounts of fatty acid methyl esters present in PL and TG were determined relative to the known amount of added 17 : 0.
HCV RNA quantification.
RNA was extracted from 150 µl serum, 10 µl low-density fraction or purified LVPs with a QIAamp viral RNA kit (Qiagen); RNA was eluted in 50 µl water and stored at –80 °C. HCV RNA quantification was performed by real-time PCR of the 5' HCV non-coding region as described previously, but with minor modifications (Komurian-Pradel et al., 2001). Briefly, RNA (4 µl) was reverse-transcribed with a Thermoscript reverse transcriptase kit (Gibco-BRL) with the RC21 primer (Besnard & Andre, 1994). Real-time PCR was carried out with 2 µl cDNA and the RC1 and RC21 primers by using an LC FastStart DNA Master SYBR Green I kit and a LightCycler apparatus (Roche Diagnostics). The proportion of HCV RNA in low-density fractions was defined as described previously (André et al., 2002).
Western blotting.
Fifteen microlitres of purified LVPs and 15 µl 100-fold-diluted low-density fraction were denaturated in Laemmli buffer and separated on a 5 % (apoB) or 10 % (E1 and E2) acrylamide gel. apoB100 and apoB48, used as controls of migration, were obtained respectively from LDLs and chylomicrons isolated from healthy plasma donors. Briefly, for apoB100, plasma density was adjusted to 1.055 g ml–1 with NaBr and ultracentrifuged as described above. Fifteen microlitres of 100-fold-diluted low-density fraction was denaturated in Laemmli buffer and loaded on the gel. For apoB48, a post-prandial plasma sample from one healthy volunteer was ultracentrifuged immediately for 4 h at 4 °C and 543 000 g with a TL100 (Beckman Instruments). Twenty microlitres of 20-fold-diluted apoB48-rich very-low-density chylomicron fraction was denaturated in Laemmli buffer and loaded on the gel. After migration, proteins were electrotransferred onto an Immobilon P membrane (Millipore). Membranes were incubated in blocking solution [20 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0.05 % Tween 20, 5 % skimmed milk] overnight at 4 °C. All following steps were performed in TBS/Tween 0.05 % at room temperature. After washing, blots were incubated for 90 min with 1D1 anti-human apoB mAb (1/10 000) or with anti-E1 (A4) or E2 (H52) (1/1000). After washing, membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse antibody (1/5000; Perbio Science). Immunoreactive proteins were visualized by using the ECL detection system (Amersham Biosciences) or SuperSignal FEMTO system (Perbio Science) and Biomax MR-film (Kodak). Bands were quantified with a videodensitometric software analyser (Imagemaster; Amersham Biosciences).
HCV envelope ELISA.
Ninety-six-well ELISA plates (Nunc MaxiSorp) were coated with 10 ng protein A per well (Sigma) in 100 µl PBS overnight at 4 °C. Plates were washed three times with washing buffer [PBS containing 0.02 % (v/v) Tween 20] and non-specific binding was blocked by addition of 200 µl blocking buffer [PBS containing 2 % (w/v) BSA] per well for 2 h at 37 °C. Low-density fractions (d<1.055 g ml–1), prepared as described in the low-density fraction preparation section above from infected (patient) or non-infected (control) plasma, were diluted from 10 to 0 µg protein ml–1 in PBS (10, 5, 3.33, 2.5, 1.67, 1.25, 0.63 and 0 µg). One hundred microlitres of each dilution was transferred to the protein A-coated plate and incubated for 60 min at 37 °C. After washing, free protein A binding sites were blocked by 150 µl human IgG (2.5 µg ml–1) for 2 h at 37 °C. An additional wash was performed and 100 µl monoclonal anti-E1 A4 (upper panel), anti-E2 H47 (lower panel) or anti-measles virus H protein (clone 55) (negative control) antiserum (diluted 1/1000 in PBS) was added to each well and incubated at 37 °C for 1 h. A further wash step was performed and 100 µl alkaline phosphatase-conjugated anti-mouse IgG (A2429; Sigma) diluted 1/1500 in PBS was incubated in each well for 1 h at 37 °C. After a final wash step, 100 µl of a 2 mg PNPP ml–1 (N2765; Sigma) substrate solution was added to each well and developed for 45 min. A405 was read.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast, apoB100 and -48 were represented equally in LVPs purified from the same whole fraction. Considering that apoB is a non-exchangeable apolipoprotein, these data indicated that viral low-density particles are associated preferentially with intestine-derived lipoproteins. For the eight patients studied (Table 1
), the proportion of plasma HCV RNA found in the <1.055 g ml–1 fraction varied from patient to patient from 10 to 95 % (mean 39 %), accordingly with a previously report (André et al., 2002). Therefore, we could estimate that 5–45 % (mean 18 %) of the total viral load in the plasma of these patients was in the form of chylomicron-like particles, considering that apoB48 was found in half of purified LVPs. The presence of apoE, -CII and -CIII, but not of apoAII, in purified LVPs further supported the TRL-like nature of these particles (Fig. 2), whereas their viral nature was confirmed by Western blot experiments with anti-envelope antibodies showing the presence of both E1 and E2 glycoproteins in purified particles (Fig. 3a). In addition, anti-envelope antibodies recognized native LVPs captured by protein A in an ELISA assay, indicating that E1 and E2 viral glycoproteins are localized at the surface of the particle (Fig. 3b).
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). The TG/apoB and PL/apoB ratios of purified LVPs increased significantly in 90 min (Table 2). By contrast, the TG/PL ratio did not significantly differ in the inter- and post-prandial periods, indicating that the TG and PL contents of the particle increased in the same proportion. Moreover, the TG/apoB mass ratios in both the pre-prandial and post-prandial periods were largely higher in LVPs than in the d<1.055 g ml–1 fraction from which LVPs were purified (Fig. 4a), indicating that LVPs are TG-enriched circulating particles in plasma. The fatty acid composition of TG and PL in purified LVPs and in the d<1.055 g ml–1 fraction was similar in the pre-prandial and in the post-prandial periods (Table 3), and very similar between the two periods (Fig. 5). These results confirmed the lipoprotein nature of LVPs. The rapid and dramatic post-prandial changes observed in the composition of LVPs while the composition of the corresponding whole d<1.055 g l–1 fraction remained steady further suggested an active contribution of the intestine to LVP production.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), leading to a protein lacking the C-terminal end of the complete apoB100 molecule. As a result, there is no direct way to capture apoB48-containing lipoproteins and therefore to demonstrate directly the association of HCV RNA with apoB48-containing lipoproteins. Despite this limitation, the strong apoB48 enrichment in protein A-captured, HCV RNA-positive LVPs compared with the plasma lipoproteins strongly suggests a direct association of HCV RNA with apoB48-containing lipoprotein. The rapid and dramatic increase in TG of these purified LVPs after lipid ingestion further strengthens the HCV–apoB48 association. These results raise the question of the nature, origin and functions of such particles.
The strong relationship between apoB-containing lipoproteins and viral particles is a specificity of HCV and related viruses (Sato et al., 1996). Although ultrastructural analysis of LVPs is necessary, these data suggest that LVPs are TRL-like particles in which the two hydrophobic domains of the core protein could be embedded in the neutral lipids of the lipoprotein core (Hope & McLauchlan, 2000; McLauchlan et al., 2002). Glycoproteins E1 and E2 may display an amphipathic helix conformation (Charloteaux et al., 2002) as apolipoproteins and insert into the surface layer of the particle. With respect to apoE, which is borne by LVPs, it has recently been shown that the E2,E3 and E2,E4 genotypes were associated respectively with a significant three- and fivefold reduction in the risk of chronic HCV infection compared with E3E4 or E3 and E4 homozygotes (Price et al., 2006). In addition, the E2,E2 genotype was never found in HCV-positive patients. The E2 isoform of apoE binds poorly to the LDL receptor (Mahley & Rall, 2000). As LVP binding to cells can be blocked by anti-apoE antibody (Agnello et al., 1999; André et al., 2002), it is likely that the defective binding of the apoE2 isoform could result in a poor uptake of LVPs. Moreover, these data support a biological role for LVPs, which, like TRLs, may have their fate and their site of clearance directed by their apolipoprotein composition (Field & Mathur, 1995).
Several mechanisms could be involved in the production of LVPs. First, LVPs could be formed within the blood circulation by the association of mature HCV virions with circulating TRLs. However, a recent study reported that HCV RNA quasispecies found in LVPs corresponded to a subgroup of the whole plasma viral population (Deforges et al., 2004). This indicates, at least, that LVPs are not issued from a random fusion of circulating HCV virions with plasma lipoproteins. Although natural antibodies against LVPs may introduce some bias in selecting a particular LVP subpopulation, the most likely hypothesis is that LVPs are formed within the endoplasmic reticulum of lipoprotein-secreting cells, in which apoB and TG are assembled to form TRLs. Indeed, immunoprecipitation of TRLs with an anti-apoB antibody precipitated 50 % of HCV RNA from HCV-infected liver macerate, indicating that a substantial amount of HCV RNA was already associated with apoB in hepatocytes (Nielsen et al., 2004). Altogether, these studies suggest that HCV association with apoB-containing lipoproteins probably occurs within lipoprotein-secreting cells, rather than resulting from binding of HCV to TRLs in the circulation.
Therefore, one should consider the hypothesis of an intestinal production of LVPs, based on the association of HCV RNA and envelope glycoproteins with apoB48-containing TRLs. Indeed, the expression of Apobec1, the editing enzyme of the apoB mRNA leading to apoB48 synthesis, is strictly restricted to enterocytes (Patterson et al., 2003) and HCV infection has not been reported to induce Apobec1 expression in hepatocytes (Jacobs et al., 2005; Smith et al., 2003; Su et al., 2002). This hypothesis is further supported by the variation in the lipid enrichment of circulating LVPs between the pre- and the post-prandial period of the patient, as expected for intestinal TRLs after food intake (Field & Mathur, 1995). Such a hypothesis is consistent with a previous study reporting that the quasispecies populations of LVPs and liver HCV RNA did not match completely, suggesting a second reservoir beside the liver and with the presence of HCV proteins in enterocytes of chronically infected patients (Deforges et al., 2004). Further investigations of chronically infected patients for detection of HCV RNA in intestinal biopsies and comparative quasispecies analysis between gut, LVPs and plasma are necessary to quantify precisely the contribution of enterocytes to the circulating viral load.
Besides the fundamental challenge to decipher the mechanisms leading to the production of LVPs, considering the intestine as a reservoir and replication site of HCV in the form of LVPs has important pathophysiological consequences. The proportion of intestinal LVPs might be substantial (mean calculated value, 18 % of the plasma viral load). As the final destination of intestinal lipoprotein remnants is the liver (Field & Mathur, 1995), an intriguing possibility could be a permanent inoculation of the liver with LVPs from the intestine. Binding and internalization of naturally antibody-coated LVPs was shown to be mediated by lipoprotein receptors that recognize apolipoproteins on the viral particles (André et al., 2002). Neutralizing antibodies directed to the envelope glycoproteins may therefore not be sufficient to control infection of the liver by LVPs. Therefore, classical virions, like those produced in vitro, and LVPs could deliver the virus with the possibility to infect the host both acutely and chronically, a feature not achieved by other flaviviruses.
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ACKNOWLEDGEMENTS |
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Received 15 March 2006;
accepted 6 June 2006.
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) or non-infected (
) patients was revealed by anti-E1 A4 (i) or anti-E2 H47 (ii). As a control, the d<1.055 g ml–1 fraction from infected (
) or non-infected (
) patients, stained by anti-H measles clone 55, is presented. Results are means of duplicates (x). Note that protein A-captured LVPs from infected patients were only recognized by anti-HCV E1 and E2 envelope antibodies and not by anti-measles virus H envelope antibodies. No material from the non-infected control was recognized by any antibody.
