1 INSERM U503, IFR128 Biosciences Lyon Gerland, 21 avenue Tony Garnier, 69365 Lyon cedex 07, France
2 UMR 2142 CNRS-bioMérieux, IFR128 Biosciences Lyon Gerland, 21 avenue Tony Garnier, 69365 Lyon cedex 07, France
3 Laboratoire d'Anatomo-Pathologie, Hôpital E. Herriot, 5 place Arsonval, 69003 Lyon, France
Correspondence
Patrice André
andre{at}cervi-lyon.inserm.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences described in this article are AY600628AY600858.
A figure showing how mAb 4F3H2 stains NS5A-expressing cells is available from JGV Online.
These authors contributed equally to this work.
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INTRODUCTION |
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Several reports have revealed the density heterogeneity of ill-defined HCV RNA-containing particles. HCV RNA-containing particles were found at density of between 1·03 and 1·25 g ml1 in the serum of chronically infected individuals (Miyamoto et al., 1992; Prince et al., 1996
; Thomssen et al., 1993
). Titration of infectivity in chimpanzee established a relationship between density of particles and infectivity, the highest infectivity of plasma being associated with the majority of HCV RNA in low-density fractions (d<1·06 g ml1), while HCV RNA found in higher density fractions appeared to be poorly infectious (Bradley et al., 1991
; Hijikata et al., 1993
). The unusually low density of some HCV RNA-containing particles suggested an association of the virus with plasma lipoproteins (Thomssen et al., 1993
). Low-density lipoproteins (LDL, d<1·06 g ml1) are particles which consist of a hydrophobic core of neutral lipid surrounded by a monolayer of amphipathic phospholipids and free cholesterol in which apolipoproteins reside (Fisher & Ginsberg, 2002
; Rustaeus et al., 1999
). Hepatocytes produce and secrete very low-density lipoprotein (VLDL) containing apolipoproteins B and E (apoB and apoE). Transformation of VLDL in the circulation gives rise to particles of smaller size, with intermediate to low density (intermediate-density lipoprotein, IDL and LDL; Fisher & Ginsberg, 2002
). Chylomicrons are another form of circulating VLDL that are secreted by intestinal epithelial cells: they resemble VLDL but contain truncated apoB, are larger, and are enriched in triglyceride. Transformation of chylomicrons in the circulation also leads to particles of smaller size and higher density (Hussain et al., 1996
; Yu & Cooper, 2001
). In an attempt to understand better the relationship between HCV and lipoprotein metabolism and to unveil specific factors required for virus replication and assembly, we recently conducted a study of the nature and infectivity of HCV particles in the low-density plasma fractions (Andre et al., 2002
). Low-density HCV RNA-containing particles (lipo-viro-particles, LVP) were rich in triglycerides, contained at least apoB, HCV RNA and core protein, and appeared as large spherical particles over 100 nm in diameter with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. These findings suggested that LVP synthesis could occur in organs specializing in production of apoB-containing lipoproteins. Because HCV replicates in the liver, this organ is probably an important source of LVP. Alternatively, because of the very high triglyceride content of LVP, intestine may also contribute to the production of these particles. To address this question we conducted a study to identify the site of LVP production.
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METHODS |
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Small intestine and liver biopsies.
Intestinal and liver biopsies were conserved at the Laboratoire d'Anatomie Pathologique, centre hospitalier Edouard Herriot, Lyon, France. Formalin-fixed liver biopsies from one HCV-seronegative patient and from four HCV RNA-positive patients were selected as negative and positive controls. Formalin-fixed and paraffin-embedded biopsies of the duodenum from 12 HCV-seropositive patients (for one patient a biopsy of the jejunum was also available) and from 12 HCV-seronegative patients were studied. Intestinal biopsies were taken for diagnostic purposes during endoscopic examinations performed for symptoms not related to HCV infection: dyspepsia in five cases, abdominal pain in five cases or suspicion of malabsorption in one case, melaena (one case) or evaluation of HCV-related vascularitis (one case). In all but two cases, the histological status of biopsy samples was normal. Aspergillosis was detected in one patient after liver transplantation for HCV-related cirrhosis, and chronic non-specific duodenitis was observed for the other patient. Ten of the 12 HCV-seropositive patients were also HCV RNA-positive by the VERSANT HCV RNA Qualitative Assay (TMA) (Bayer). For RNA extraction, biopsies were sliced into small pieces, deparaffinated and rehydrated as described below for immunostaining. Then 150 µl of 6 mg ml1 proteinase K (Sigma) complemented with 20 µg yeast tRNA was added to the sections, and digestion was performed overnight at 45 °C. Total RNA was further extracted with the NucleoSpin RNA virus kit and eluted in 50 µl RNase-free water.
Preparation of low-density fractions.
Plasma from infected patients was separated by ultracentrifugation to obtain one low-density fraction composed of the apoB-containing lipoproteins including chylomicrons, VLDL, IDL and LDL. Density of the plasma was adjusted to d=1·055 g ml1 with NaBr (Sigma). The low-density fraction (d<1·055 g ml1) was obtained by centrifugation of plasma for 4 h at 4 °C and 543 000 g with the TLA100.4 rotor and TL100 ultracentrifuge (Beckman Instruments). The top fraction was collected and extensively dialysed at 4 °C against 150 mM NaCl, 0·24 mM EDTA pH 7·4 buffer, filtered through 0·22 µm filters (Millipore) and stored at 4 °C in the dark.
Purification of LVP.
This procedure (Andre et al., 2002) has been extensively described and leads to the purification of HCV LVP. Briefly, 10 µl Protein A-coated magnetic beads (Miltenyi Biotec) were incubated at room temperature with 1 ml of the low-density fractions in PBS with gentle rocking for 30 min. Beads were then passed through a magnetic column (Miltenyi Biotec), washed and collected in 500 µl PBS or DMEM/0·2 % BSA (Gibco-BRL). Samples with high lipid content were first diluted with 2 vols of normal human serum to inactivate PCR inhibitors, and RNA was extracted from 10 µl LVP with the NucleoSpin RNA virus kit, eluted in 50 µl and stored at 80 °C.
HCV RNA quantification.
HCV positive-strand RNA quantification was performed by real-time PCR in the 5' HCV non-coding region as described previously, with minor modifications (Komurian-Pradel et al., 2001). Briefly, RNA (4 µl) was reverse-transcribed with the Thermoscript Reverse transcriptase kit (Gibco-BRL) using the RC21 primer. Real-time PCR was carried out with 2 µl cDNA and RC1 and RC21 primers using the LC FastStart DNA Master SYBR Green I kit and the LightCycler apparatus (Roche Diagnostics).
An index of HCV RNA association with low-density fractions was determined including apoB as an internal standard of the lipoprotein compartment (Andre et al., 2002). The apoB concentration in fractions and sera was determined using an immunochemical kit following the manufacturer's procedure (Apo B kit; bioMérieux). The concentration was determined from a calibration curve established with the Apo B kit standard.
The index of HCV RNA association with LDF was calculated as follows:
(RNA copy number per mg apoB in LDF/RNA copy number per mg apoB in serum)x100
Quantification of negative-strand HCV RNA was performed following the same procedure, except that a tag-RC1 primer was used instead of RC1 to initiate the reverse transcription (5'-ggc cgt cat ggt ggc gaa taa GTC TAG CCA TGG CGT TAG TA-3'), and the tag was used as primer during the amplification reaction. Details of the negative-strand quantification method are described elsewhere (Komurian-Pradel et al., 2004).
Sequencing and genotyping of the 5' HCV non-coding region.
PCR amplification products that were synthesized during the HCV RNA quantification process with the LightCycler were sequenced directly in both directions using the PRISM Ready Reaction AmpliTaq FS BigDye Terminator Cycle Sequencing Kit (PE/Applied Biosystems) with the Applied Biosystem 377 and 373A automated DNA sequencers. The HCV genotype was determined by comparison of nucleotide sequences with those of the Hepatitis C Virus DataBase (http://hepatitis.ibcp.fr).
Cloning of the virus quasispecies.
The HCV core and envelope 2 (E2) regions were targeted for amplification by nested RT-PCR. Virus RNA (8 µl) was reverse-transcribed in a volume of 20 µl using the antisense primers 5'-CAT CAT ATC CCA AGC CAT-3' for core and 5'-ACG GTC GAG GTG CGT ART GC-3' for E2 genes, then amplified for 35 cycles (denaturation at 94 °C for 45 s, hybridization at 55 °C for 30 s and primer extension at 72 °C for 60 s) using the sense primers 5'-GCT TGC GAG TGC CCC GGG AGG TCT-3' and 5'-GTA ACA GGT CAC CGC ATG GC-3' for core and E2 genes, respectively. One-tenth of the volume of the first PCR products was re-amplified for 35 cycles with internal primers 5'-ATG AGC ACG AAT CCT AAA CC-3' and 5'-GGT ATC GAT GAC CTT ACC CA-3' for core gene and 5'-GCA TGG CTT GGG ATA TGA TG-3' and 5'-GCA GTC CTG TTG ATG TGC CA-3' for E2 gene. Fragments 375 and 285 bp, respectively, were obtained for the core and E2 genes, which includes the hypervariable region 1 (HVRI). The amplified PCR products of core and E2 regions were purified using the QIAquick PCR purification kit (Qiagen), cloned into the TOPO TA Cloning Kit and transformed into Escherichia coli strain One Shot TOPO 10 competent cells (Invitrogen). At least 10 clones were selected for each individual and sample. Plasmid DNA was extracted with the Qiagen plasmid kit (QIAprep Miniprep; Qiagen) and sequenced using the PRISM Ready Reaction AmpliTaqFS BigDye Terminator Cycle Sequencing Kit and ABI PRISM 3100 Genetic Analyser (PE/Applied Biosystems).
Quasispecies sequence analysis.
Nucleotide sequences were aligned using CLUSTAL W 1.74 (Thompson et al., 1994) and refined by visual inspection with SEAVIEW (Galtier et al., 1996
). DNA distance matrix and phylogenetic trees were computed with PHYLO_WIN (Galtier et al., 1996
). Distances between sequences were computed under the Kimura two-parameter model (Kimura, 1980
). Trees were built using the neighbour-joining method (Saitou & Nei, 1987
) and tree topologies were tested with 1000 bootstrap sampling replicates.
In order to determine whether sequences from a given compartment shared more genetic identity with each other than with sequences from other compartments, we used Mantel's test (Mantel, 1967). This test was performed using ADE-4 (Thioulouse, 1989
). The method consists of comparing a DNA distance matrix to a compartmentalized reference distribution matrix of the same dimensions, where the (i,j) value of the matrix is set to 0 if sequence i is from the same compartment as sequence j and the (i,j) value is set to 1 in the other case. The Pearson correlation coefficient r2 was computed for all pairs (observed r2). The null distribution was constructed by permuting the rows and columns of the reference matrix 10 000 times. From this distribution, the number of times when the observed r2 was exceeded gave the exact P value of the correlation observed.
Normalized Shannon entropies were calculated as described (Roque Afonso et al., 1999; Wolinsky et al., 1996
). Differences between compartments' genetic distances were assessed using the non-parametric MannWhitney test (STATVIEW II; Abacus Concept, Berkeley, CA, USA).
Immunostaining of biopsies.
Sections of formalin-fixed paraffin-embedded biopsies of the liver and small intestine were deparaffinated and rehydrated in two baths of methylcyclohexane, two baths of 100 % ethanol, one bath of 70 % ethanol and one bath of PBS for 10 min each. Sections were placed in 0·01 M citrate buffer pH 6·0, treated for 16 min in a microwave oven at 650 W and then allowed to cool slowly to room temperature. Endogenous peroxidase was inhibited by incubation with 1 % H2O2 in PBS for 10 min, and sections were placed in PBS/0·2 % BSA for 30 min. mAbs to HCV NS3 (clone 4G10H4), NS5a (clone 4F3H2) and an irrelevant mAb (clone 17D1C11) were provided by bioMérieux and were of isotype IgG1. They were applied for 1 h at a final concentration of 0·07 mg ml1 at room temperature. After four washes in PBS/0·2 % BSA, sections were incubated with Envision+System HRP Mouse (Dako) for 45 min. Following four washes in PBS/0·2 % BSA and two washes in PBS, staining was developed for 10 min with a DAB peroxidase substrate kit (Vector). Sections were counterstained with Mayer's haematoxylin (Dako) and mounted in 90 % glycerol. Images were recorded with a Sony Power HAD digital camera. Monoclonal anti-NS5A antibody 4F3H2 was tested and titrated on cells stably transfected and expressing NS5A provided by D. Moradpour (Polyak et al., 1999) (see supplementary figure in JGV Online).
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RESULTS |
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DISCUSSION |
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LVP enter hepatoma cell lines through the LDL receptor, which recognizes apoB and apoE on the particle. Binding of purified LVP to HepG2 cells is very efficient and is competed out by native lipoproteins, explaining the poor binding of non-purified HCV from infected serum (Andre et al., 2002). The LDL receptor is expressed in vivo by intestinal cells (Levy et al., 2000
). The entry route in enterocytes is therefore likely to be similar to that of hepatocytes. However, besides the LDL receptor, CD81 and the scavenger receptor B class 1 (SR-B1) have been described as putative receptors for the viral envelope proteins (Pileri et al., 1998
; Scarselli et al., 2002
). CD81 is not expressed in the intestine (Okochi et al., 1999
), but SR-B1 was detected at the apical and basal poles of enterocytes and could be an alternative entry pathway for E1/E2-containing virus particles (Altmann et al., 2002
; Cai et al., 2001
). However, analysis of LVP quasispecies showed that the binding site of E2 (HVR1) to SR-B1 does not appear to be subjected to any selection. It is thus likely that no constraint is exerted by this receptor on the envelope protein, and that LVP do not predominantly enter the intestinal cells via SR-B1.
Thinking of the intestine as a reservoir for HCV-producing low-density infectious virus particles modifies the current understanding of the pathogenesis of HCV infection and raises several questions. First, HCV infection of small intestine was, surprisingly, not accompanied by inflammatory response, as mild duodenitis was observed in only one case. We recently showed that the lipid composition of apoB-containing lipoproteins and of lipid emulsions has important consequences for dendritic cell maturation and function (Coutant et al., 2004; Perrin-Cocon et al., 2001
). It is conceivable that the lipid moiety of the low-density HCV particles induces a state of immune unresponsiveness. The mechanism of such inhibition is under investigation. Second, because most chylomicrons and chylomicron remnants are captured by hepatocytes (Hussain et al., 1996
; Yu & Cooper, 2001
), small intestine might provide a source of infectious particles responsible for continuous liver infection and for infection of liver grafts. Such a mechanism would have important consequences for antiviral therapy. Efficient drugs should target intestinal cells, and analysis of drug metabolism in these cells would be an important step in the development of antiviral therapy. Third, infection of intestinal cells in vitro may provide opportunities to understand virus replication and assembly better. The Caco-2 cell line is used as a model of human intestinal cells to study apoB metabolism and lipoprotein synthesis (Levy et al., 1995
). The possibility of inducing the differentiation of Caco-2 in vitro and modulating lipoprotein secretion should allow a detailed analysis of the relationship between LVP and lipoprotein assembly, leading to a cell-based HCV replication system. Synthesis of chylomicron and VLDL depends on apoB synthesis and on its translocation into the lumen of the endoplasmic reticulum. If nascent lipoproteins are not loaded with triglyceride, apoB is retro-translocated into the cytoplasm where it is degraded by the proteasome (Hussain et al., 1996
; Luchoomun & Hussain, 1999
; Yu & Cooper, 2001
). Because triglycerides are synthesized by enterocytes from dietary fatty acids and because chylomicron synthesis and secretion follow lipid digestion, it would be of interest to assess the influence of starving and of lipid-rich meals on HCV viraemia. In support of this idea, a negative correlation has recently been reported between plasma apoB concentration and HCV virus load, particularly for patients infected with genotype non-1 (Petit et al., 2003
).
In conclusion, the presence of HCV proteins in enterocytes further emphasizes the interaction between lipoprotein metabolism and HCV, and modifies our current understanding of hepatitis C.
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ACKNOWLEDGEMENTS |
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Received 28 February 2004;
accepted 14 April 2004.
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