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Journal of Virology logoLink to Journal of Virology
. 2015 Apr 22;89(13):6782–6791. doi: 10.1128/JVI.00194-15

The Serum Very-Low-Density Lipoprotein Serves as a Restriction Factor against Hepatitis C Virus Infection

Jian Tao a,b, Kyung-Don Kang b, Stacy D Hall b, Audra H Laube c, Jia Liu a, Matthew B Renfrow c, Jan Novak b, Guangxiang Luo a,b,✉
Editor: J-H J Ou
PMCID: PMC4468503  PMID: 25903344

ABSTRACT

Recent studies demonstrated that transgenic mice expressing key human hepatitis C virus (HCV) receptors are susceptible to HCV infection, albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or the presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effects of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. The results also imply that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication.

IMPORTANCE HCV is a major cause of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in the mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines.

INTRODUCTION

Hepatitis C virus (HCV) is an enveloped RNA virus containing a single-stranded and positive-sense RNA genome of 9.6 kb in length. It is the prototype of the Hepacivirus genus in the Flaviviridae family. The virion RNA (vRNA) genome encodes a large polyprotein precursor that is proteolytically cleaved by cellular and viral proteases into structural (core, E1, E2, and p7) and nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The viral structural proteins C, E1, and E2 are sufficient for the formation of virus-like particles (1), while NS3, NS4A, NS4B, NS5A, and NS5B are the minimal set of viral proteins essential for RNA replication (2). HCV enters cells via receptor-mediated endocytosis. The cellular protein apolipoprotein E (apoE) incorporated onto the HCV envelope mediates its attachment via binding to the cell-surface heparan sulfate proteoglycans (HSPGs) (3, 4). Other cell-surface receptors or coreceptors, including CD81, claudin, occludin, low-density lipoprotein receptor (LDLR), and SR-BI, mainly act at postattachment steps through specific interactions with the viral envelope glycoproteins E1 and E2 to promote HCV cell entry (5–7). Upon internalization and uncoating, HCV RNA genome initially serves as an mRNA for viral polyprotein translation and then as a template for negative-strand RNA synthesis. Viral RNA replication occurs in the endoplasmic reticulum (ER) membrane-associated replication complex consisting of viral NS proteins and many cellular proteins (8). Progeny HCV particles are formed in the lipid droplet-associated membrane structures, maturated through the trans-Golgi network, and finally egressed from the infected cells (9, 10).

A great deal of new knowledge with respect to the details of the HCV life cycle in vitro has recently been obtained (11, 12), using newly developed infectious HCV cell culture models (13–16). However, little is known about the underlying mechanisms of viral pathogenesis and carcinogenesis, host response to HCV infection, and virus-host interaction in vivo primarily due to the lack of small animal models of HCV infection and replication (17). The recent development of humanized mice and transgenic mice expressing key human HCV receptors (18, 19), which are susceptible to HCV infection, holds a great promise to recapitulate the entire HCV life cycle in vivo. For unknown reasons, HCV infection and replication in the humanized mice and transgenic mice were very inefficient (18–21), suggesting possible host factors that restrict HCV infection and/or replication.

In the present study, we sought to determine whether serum contains a restriction factor or factors that inhibit HCV infection. Notably, both mouse and human sera were found to remarkably inhibit HCV infection in cell culture. Results from time-of-addition experiments demonstrate that serum restriction factors blocked HCV attachment to the cell surface. More importantly, the very-low-density lipoprotein (VLDL) particles present in the serum were identified as restriction factors of HCV infection. These findings suggest that VLDL in the patients' plasma is inhibitory to HCV infection and spread. Thus, it is possible that elimination of VLDL from the plasma of humanized mice will likely facilitate the development of more robust mouse models for the study of HCV pathogenesis, carcinogenesis, and virus-host interaction, as well as the development of effective HCV vaccines.

MATERIALS AND METHODS

Cell culture and HCV.

The highly HCV-permissive human hepatoma cell line (Huh-7.5) was provided by Charles M. Rice and Apath, LLC (22). Huh-7.5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Benchmark), 0.1 mM nonessential amino acids (Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C in a 5% CO2 incubator. The cell culture-adapted HCV of genotype 2a (JFH1) was grown in Huh-7.5 cells and cleared by filtration through a 0.22-μm-pore filter unit (Corning), as previously described (23).

Antibodies and sera.

HCV NS5A monoclonal antibody (9E10) was generously provided by Charles M. Rice. β-Actin monoclonal antibody (AC15) was purchased from Sigma-Aldrich. Goat anti-mouse secondary antibody conjugated with HRP was from Invitrogen; apoB antibody was from Biodesign. HCV NS3 monoclonal antibody (mAb15) was produced in the laboratory as previously described (24). Mouse serum was obtained by bleeding 8- to 12-week-old BALB/cJ mice (The Jackson Laboratory). Human serum was purchased from Sigma-Aldrich. Purified human VLDL was from Meridian.

HCV attachment and infection assays.

Cell culture plates were coated with 50 μg/ml of collagen at 37°C for 30 min. Huh-7.5 cells were seeded overnight at 37°C. For the HCV attachment assay, Huh-7.5 cells in 24-well cell culture plates were infected with HCV at a multiplicity of infection (MOI) of 10 in the presence of increasing concentrations of mouse serum on ice for 2 h. The unbound virus was removed by washing with 1× phosphate-buffered saline (PBS) three times. The vRNA of cell-bound HCV was extracted with RNAzol reagent (Molecular Research Center) and was used for quantification of vRNA by a quantitative reverse transcription-PCR (qRT-PCR) method using the mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. HCV infection was carried out by incubation of Huh-7.5 cells with HCV in the presence of various concentrations of mouse and human sera or purified mouse and human VLDL at 37°C for 2 h. The HCV-infected cells with and without serum or VLDL treatment were washed with PBS and then incubated with fresh Dulbecco's modified Eagle's medium (DMEM) for 24 h (single-cycle HCV growth). The cell culture supernatants were collected for determination of infectious HCV titers by limiting dilution and immunofluorescence assay (IFA). For detection of viral proteins, the HCV-infected Huh-7.5 cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 150 mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate). Total RNA was extracted from the HCV-infected cells with RNAzol reagent and was used for quantification of HCV RNA by qRT-PCR. HCV RNA was quantified by a real-time qRT-PCR method using GAPDH mRNA as an internal control (25).

WB analysis.

For Western blot (WB) analysis, the protein concentration of cell lysates was determined using a protein assay reagent (Bio-Rad). Twenty micrograms of total protein for each sample was electrophoresed in a 10% sodium dodecyl sulfate–polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% dry milk, the membrane was incubated with primary antibodies specific to NS5A, NS3, apoB, and β-actin and then with secondary anti-mouse antibody conjugated with peroxidase. Proteins were visualized by chemiluminescence (ECL) staining with a Bio-Rad ChemiDoc MP Imaging System (3).

RT-PCR.

The levels of HCV vRNA and positive-strand RNA in the infected cells were determined by a quantitative one-step RT-PCR method using specific primers and probes. Oligonucleotide primers 2a-F (5′-AGCCATGGCGTTAGTATGAGTGTC-3′) and 2a-R (5′-ACAAGGCCTTTCGCAACCCAA-3′) and the TaqMan probe (5′-AAACCCACTCTATGCCCGGCCATTT-3′) are complementary to the 5′ untranscribed region (UTR) sequences (IDT). The one-step real-time qRT-PCR was done using the following program: 50°C for 10 min and 95°C for 5 min followed by 40 cycles at 95°C for 10 s and 60°C for 30s. GAPDH mRNA was used as an internal control for normalization using the primer and probe set from Applied Biosystems.

IFA.

Infectious HCV titers in cell culture supernatants were determined by 10-fold serial dilution and IFA. Serially diluted HCV was used to infect Huh-7.5 cells in 96-well plates. After 2 h of incubation at 37°C, 100 μl fresh DMEM containing 1% methylcellulose was added into each well. At 48 h postinfection (p.i.), focus-forming units (FFU) per milliliter were determined by IFA staining as previously described (26). Briefly, the HCV-infected cells were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X-100. An NS3-specific monoclonal antibody was used to stain the virus-infected cell foci, which were visualized with the secondary donkey anti-mouse IgG conjugated with Alexa Fluor 594 dye (Molecular Probes). Infectious HCV titers (FFU per milliliter) were calculated based on the average numbers of NS3-positive foci in triplicates.

Size exclusion chromatography and targeted MS analyses.

To remove insoluble materials, mouse serum was centrifuged at 12,000 rpm for 10 min and then passaged through a 0.22-μm-pore filter unit. Two hundred microliters of clarified mouse serum was applied to a calibrated Superose 6 10/300GL column. Serum was eluted into 100 fractions (covering the range of molecules from the void volume to 1 kDa; 0.25/ml/fraction) with PBS at a flow rate of 0.3 ml/min, following the previously described procedures (27). The inhibitory activity of each fraction was determined by a single-cycle HCV growth assay, as described above in the HCV infection assay. To determine proteins uniquely present in the active fractions, active fractions and adjacent inactive fractions were analyzed by high-resolution mass spectrometry. Briefly, active fractions were pooled and fractionated by SDS-PAGE under reducing conditions. Individual protein bands were excised from the gel and subjected to in-gel tryptic digestion (reduced with 10 mM dithiothreitol and alkylated with 50 mM iodoacetamide). Digested alkylated peptides were loaded onto a self-prepared 11-cm, 100-μm-diameter pulled tip packed with Jupiter 5-μm C18 reversed-phase beads (Phenomenex, Torrance, CA). Samples were analytically separated via nano-liquid chromatography (nano-LC) by use of an Eksigent MicroAS autosampler and two-dimensional LC nanopump (Eksigent, Dublin, CA). Tryptic peptides were separated by liquid chromatography using a gradient of acetonitrile containing 0.2% formic acid, and the eluted tryptic peptides were electrosprayed at 2 kV into a dual linear quadrupole ion trap Orbitrap Velos Pro mass spectrometer (Orbitrap MS; Thermo Fisher Scientific, San Jose, CA). Peptides were analyzed making use of collision-induced dissociation (CID) for the tandem mass spectrometry (MS/MS) scans. The mass spectrometer was set to switch between MS/MS scans of the 18 most abundant precursor ions. The dynamic exclusion setting was set to exclude ions for 2 min after a repeat count of 3 for a duration of 45 s. Thermo Xcalibur Raw files were converted to mzXML files using the converter ReAdW program. For identification of proteins and peptides, the search engine TurboSEQUEST v.27 (rev.12; Thermo, Fisher Scientific) was utilized with parent-ion mass accuracy of 10.0 ppm with the unimouse database. SEQUEST searches were processed and visualized using Scaffold 3.0 (Proteome Software, Inc., Portland, OR) with the addition of searching with X!Tandem on the Scaffold software with 95% confidence at the peptide level and 99% confidence at the protein level.

Isolation of VLDL from mouse serum.

Mouse VLDL was isolated using a modified ultracentrifugation method described previously (28). Briefly, mouse serum was adjusted to a density of 1.019 g/ml using potassium bromide (KBr) and then ultracentrifuged at 55,000 rpm in a fixed-angle rotor (NVT 90) for 5.5 h at 10°C. The gradient was fractionated manually from the bottom of the tube, collecting 0.5 ml for each fraction. A total of 10 fractions were collected and desalted by dilution with PBS. KBr was subsequently removed by centrifugation at 3,000 rpm for 30 min at 10°C in Millipore concentrators (molecular weight cutoff [MWCO], 3,000). The inhibitory activity of each fraction was determined using the single-cycle HCV growth assay as described above.

Administration of an inhibitor of the MTP.

Five 4-week-old C57BL/6J mice (Jackson Laboratory) were administered orally a microsomal triglyceride transfer protein (MTP) inhibitor, CP-346086 (Sigma-Aldrich), in 0.5% methylcellulose at 33 mg/kg body weight once daily for 14 days according to the protocol described previously (29). The other 5 mice were similarly treated with 0.5% methylcellulose as a vehicle control group. Mouse sera were collected by bleeding and centrifugation and used for determination of HCV-inhibitory activity and detection of apoB by Western blotting.

RESULTS

Restriction of HCV infection by mouse and human sera.

Previous studies by others demonstrated that mouse expressing key human receptors is susceptible to HCV infection, albeit very inefficiently (18, 20). The low levels of HCV replication in the hepatocytes of humanized mice could be due to restriction at HCV infection and/or replication steps. In this study, we sought to determine whether HCV infection is inhibited by a restriction factor or factors in mouse and human sera. Initially, we used an HCV single-cycle growth assay (24 h after HCV infection) to examine the effects of mouse and human sera on HCV infection. Huh-7.5 cells were infected with HCV at an MOI of 10 in the presence of increasing concentrations (0, 0.625, 1.25, 2.5, 5, and 10%) of mouse serum at 37°C for 2 h, followed by extensive washing of cells with PBS to remove unbound virus. At 24 h postinfection (p.i.), the levels of HCV protein and positive-strand viral RNA in the HCV-infected Huh-7.5 cells were determined by Western blotting and qRT-PCR, respectively. In addition, the infectious HCV titers in the cell culture supernatants were quantified by IFA upon limiting dilution. Our results revealed that mouse serum efficiently inhibited HCV infection, resulting in a remarkable decrease of both viral protein and RNA upon a single cycle of HCV growth (Fig. 1). The levels of HCV NS5A protein were proportionally reduced by increasing concentrations of mouse serum with undetectable NS5A at 5% and 10% mouse serum (Fig. 1A). Similarly, the levels of viral positive-strand RNA were decreased by mouse serum in a dose-dependent manner with a 90% reduction of HCV RNA at 10% mouse serum (Fig. 1B). Likewise, mouse serum lowered infectious HCV titers by up to 100-fold at the serum concentration of 10% (Fig. 1C). These results clearly show that mouse serum contains potent restriction factor(s) of HCV infection.

FIG 1.

FIG 1

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Effects of mouse serum on HCV infection. Huh-7.5 cells were infected with HCV in the absence or presence of increasing concentrations (0, 0.625, 1.25, 2.5, 5, and 10%) of mouse serum at 37°C for 2 h. After being washed with PBS, the HCV-infected cells were incubated with fresh DMEM at 37°C for 24 h. (A) Detection of NS5A by Western blotting (WB). NS5A in the HCV-infected cells was detected by WB using an NS5A-specific monoclonal antibody (9E10) with β-actin as an internal control. Proteins were visualized using an HRP-conjugated anti-mouse IgG, the ECL enhanced chemiluminescence substrate, and the Bio-Rad ChemiDoc MP imaging system. (B) Quantification of positive-strand HCV RNA by qRT-PCR. The levels of HCV vRNA in infected cells were quantified by a qRT-PCR method and were converted to percentage of the control, considering the HCV RNA level in the absence of mouse serum as 100%. (C) Titration of infectious HCV titers. The titers of infectious HCV in the cell culture supernatants in triplicates were determined by serial dilution and immunofluorescence staining for NS3-positive cell foci (FFU), as previously described (13, 26). Serum concentrations are indicated by the numbers at the bottom.

To determine whether human serum also inhibited HCV infection, the HCV infection experiments described above were carried out in the presence of increasing concentrations of human serum. Similar to mouse serum, human serum significantly inhibited HCV infection in a dose-dependent manner (Fig. 2). However, human serum was less potent in inhibition of HCV infection, resulting in decreases in NS5A protein and viral RNA by 80% and 60%, respectively, at a 10% concentration (Fig. 2B). Taken together, these findings suggest that mouse and human sera contain a restriction factor or factors that can block HCV infection.

FIG 2.

FIG 2

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Effects of human serum on HCV infection. Experiments were done in the same way as Fig. 1, except human serum was used. After 24 h p.i., the HCV-infected cells were lysed using a RIPA buffer. NS5A was detected by WB with β-actin as a control (A). The levels of positive-strand HCV RNA were quantified by a qRT-PCR method and were converted to a percentage of the control, considering 100% of the HCV RNA in the absence of serum (B). The numbers above the protein bands or at the bottom of the RNA graph indicate serum concentrations (percentages) used in the experiments.

Mouse serum blocked HCV attachment but not replication.

To further understand the mechanism of action of the serum restriction factors, the effects of mouse serum on HCV attachment and replication were determined. The HCV attachment assay was done based on a method used in our earlier studies (3). Huh-7.5 cells in 24-well cell culture plates were incubated with HCV in the presence of increasing concentrations of mouse serum on ice for 2 h. Upon HCV attachment to cells, the unbound virus was removed by extensive washing with PBS. The virion RNA (vRNA) of the cell-bound HCV was extracted and quantified by a real-time qRT-PCR method using primers and probe complementary to the 5′ UTR sequence. Interestingly, mouse serum potently blocked HCV cell attachment. The levels of cell-bound HCV vRNA were reduced by 61, 72, 80, 85, and 88% at serum concentrations of 0.625, 1.25, 2.5, 5, and 10%, respectively (Fig. 3A). However, mouse serum did not significantly affect HCV replication when added after HCV bound cells based on similar levels of NS5A (Fig. 3B). Collectively, these findings demonstrate that serum restriction factors inhibited HCV infection by specifically blocking its attachment to the surface of hepatocytes without any significant effect on HCV replication.

FIG 3.

FIG 3

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Effects of mouse serum on HCV cell attachment and replication. (A) Inhibition of HCV attachment by mouse serum. Huh-7.5 cells were incubated with HCV at an MOI of 10 in the presence of increasing concentrations (0, 0.625, 1.25, 2.5, 5, and 10%) of mouse serum on ice for 2 h. Upon removal of unbound virus, the vRNA of cell-bound HCV was extracted with RNAzol reagent and quantified using a one-step qRT-PCR method. Average levels of HCV vRNA from three independent experiments are plotted against mouse serum concentrations. (B) Effects of mouse serum on HCV replication. Huh-7.5 cells were infected with HCV at 37°C for 2 h without mouse serum. After extensive washing with PBS, the HCV-infected cells were incubated with fresh DMEM containing increasing concentrations (0, 0.625, 1.25, 2.5, 5, and 10%) of mouse serum at 37°C for 24 h. The levels of NS5A in the HCV-infected cells were determined by WB with β-actin as an internal control.

Separation and identification of serum restriction factors of HCV infection.

To identify potential restriction factors present in mouse serum, size exclusion chromatography was used to fractionate restriction factors in mouse serum. A total of 200 μl of mouse serum was loaded onto a calibrated Superose 6 column and separated into 100 fractions by collecting 0.25 ml of eluent in each fraction. The HCV-inhibitory activity of each fraction was then determined by the above-described single-cycle HCV growth assay. Huh-7.5 cells in 24-well cell culture plates were infected with HCV in the presence of 20% eluent from each fraction at 37°C for 2 h. At 24 h p.i., the HCV-infected Huh-7.5 cells were lysed and used for detection of HCV NS5A by Western blotting. Results indicated that fractions 30 to 51 exhibited potent activity against HCV infection, resulting in a reduction of NS5A to an undetectable level (Fig. 4A). Further titration of the active fractions 38 to 41 demonstrated a dose-dependent inhibition of HCV infection, lowering NS5A levels by 70, 95, and 100% at eluent concentrations of 0.625%, 2.5%, and 10%, respectively (Fig. 4B). In contrast, the fractions prior to fraction 30 or after fraction 51 had little or no inhibitory activity, suggesting that specific serum restriction factors are heterogeneous in size, being present in fractions 30 to 51.

FIG 4.

FIG 4

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Determination of HCV-inhibitory activity in serum fractionated by size exclusion chromatography. Mouse serum was fractioned by size exclusion chromatography on a calibrated Superose 6 column, with the 100 fractions collected representing a range of apparent molecular masses from the void volume to 10 kDa. Huh-7.5 cells were infected with HCV in the presence of 20% of each fraction at 37°C for 2 h. After a 24-h incubation, NS5A was detected by WB using β-actin as an internal control. (−), mock control; (+), HCV-infected Huh-7.5 cell lysate as a positive control. (A). Fraction numbers are indicated on the top. The inhibitory activity of active fractions (fractions 38 to 41) was further assessed by dilution from 10% to 0.625% (B).

To reveal the identities of potential restriction factor(s) present in inhibitory fractions, fraction 40, in the middle of fractions with HCV-inhibitory activity, along with adjacent inactive fractions, was chosen for mass spectrometric analysis. The active fraction contained several serum proteins, including apolipoproteins A1 (apoA1), B100 and B48 (apoB100 and apoB48), and E (apoE) (data not shown). Thus, a possible candidate for the restriction factor could be the very-low-density lipoprotein (VLDL) because our earlier studies suggested that VLDL was the most potent inhibitor of HCV infection among high-density lipoprotein (HDL), LDL, and VLDL (Z.-H. Cai and G. Luo, unpublished results). HDL also contains apoE, which determines HCV infectivity and virion assembly (24). However, the serum obtained from apoE-knockout (apoE−/−) mice had even higher HCV-inhibitory activity (data not shown). apoB is a signature apolipoprotein assembled in VLDL. To confirm the existence of VLDL in the fractions with HCV-inhibitory activity, 10 fractions, including inactive fractions 20, 23, 26, 29, 52, 56, and 60 and active fractions 34, 40, and 46, were chosen for detection of apoB by Western blotting (Fig. 5). Consistent with data from mass spectrometric analysis, apoB100 was detected in all fractions with HCV-inhibitory activity but not in the fractions without inhibitory activity (Fig. 5). Collectively, these data suggest that VLDL in mouse and human sera is the major restriction factor for the blockade of HCV infection.

FIG 5.

FIG 5

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Detection of apoB in different mouse serum fractions by WB. Three micrograms from each of the fractions 20, 23, 26, 29, 34, 40, 46, 52, 56, and 60 was separated in a 10% SDS–polyacrylamide gel. Upon transfer onto a PVDF membrane, apoB was detected by WB using a specific antibody. Purified mouse VLDL (fraction 1-F1 in Fig. 6) was used as a positive control for detection of apoB. Fraction numbers are indicated on the top.

Serum VLDL serves as the major restriction factor of HCV infection.

To validate the apoB-containing VLDL as the major restriction factor of HCV infection, a sedimentation fractionation method was used to purify VLDL from mouse serum. Upon ultracentrifugation, 10 fractions (0.5 ml each) were collected from the bottom of the tube. The inhibitory activity of each fraction was determined using a single-cycle HCV growth assay. Huh-7.5 cells in 24-well plates were infected with HCV in the presence of 5% of each fraction at 37°C for 2 h. At 24 h p.i., HCV NS3 and RNA in the HCV-infected Huh-7.5 cells were quantified by Western blotting and qRT-PCR, respectively (Fig. 6). VLDL is expected to float on the top of the gradient upon ultracentrifugation. As expected, the top fraction, containing mouse VLDL, resulted in a remarkable reduction of HCV protein to an undetectable level (Fig. 6A) and a decrease of HCV RNA by 95% (Fig. 6B). Fraction 2 modestly lowered HCV protein and RNA, suggesting some VLDL also present in this fraction. The other fractions did not have any significant inhibitory activity (Fig. 6). To further determine the potency of purified mouse VLDL for inhibition of HCV infection, fraction 1 was titrated using the single-cycle HCV growth assay (Fig. 6C and D). Purified mouse VLDL blocked HCV infection more efficiently than unfractionated mouse serum, resulting in a proportional decrease of HCV protein to an undetectable level at a 5% concentration (Fig. 6C) and also a dose-dependent reduction of viral RNA by 31, 52, and 85% at concentrations of 0.3, 1.25, and 5%, respectively (Fig. 6D).

FIG 6.

FIG 6

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Inhibition of HCV infection by purified mouse VLDL. Mouse serum VLDL was isolated by a previously described method (28). Huh-7.5 cells were infected with HCV at an MOI of 10 in the presence of 5% of each fraction at 37°C for 2 h. After 24 h p.i., the HCV-infected Huh-7.5 cells were lysed and used for detection of HCV NS3 protein by WB with β-actin as an internal control (A). The levels of positive-strand HCV RNA were quantified by a qRT-PCR method and were converted to a percentage of the control considering 100% of the HCV RNA in the presence of 5% PBS (B). Fraction 1 containing purified VLDL was titrated from 5% to 0.3% for its inhibition of HCV infection using the single-cycle HCV growth assay similar to Fig. 4B. Inhibition of HCV infection by purified mouse VLDL was determined by measuring the levels of NS3 protein by WB (C) as well as the relative levels of positive-strand HCV RNA compared to the control by qRT-PCR (D).

We also validated the HCV-inhibitory activity of purified human VLDL (Fig. 7). Similar to purified mouse VLDL, purified human VLDL efficiently inhibited HCV infection in a dose-dependent manner (Fig. 7). Both HCV protein and viral positive-strand RNA were decreased by 80% at concentrations up to 25 μg/ml of purified human VLDL (Fig. 7A and B). These findings demonstrate that both mouse VLDL and human VLDL serve as the major restriction factor of HCV infection.

FIG 7.

FIG 7

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Blockade of HCV infection by purified human VLDL. Similar to Fig. 6, inhibition of HCV infection by purified human VLDL was determined using the single-cycle HCV growth assay. Huh-7.5 cells were infected with HCV in the presence of various concentrations (0, 0.04, 0.2, 1, 5, and 25 μg/ml) of purified human VLDL at 37°C for 2 h. After 24 h p.i., the HCV-infected cells were lysed for detection of NS5A (A), or total RNA was extracted with RNAzol for quantification of positive-strand HCV RNA by qRT-PCR (B). The levels of HCV RNA were converted to a percentage of the control, considering 100% of the HCV RNA in the absence of VLDL. Average levels of HCV RNA from three independent experiments were calculated with standard deviations (SD) indicated.

The question arose of whether the blockade of VLDL assembly and secretion by using an MTP inhibitor would reduce the HCV-inhibitory activity in mouse serum. A group of 5 mice received an oral administration of MTP inhibitor CP-346086 in 0.5% methylcellulose at 33 mg/kg/day for 14 days. The control group of 5 animals was only treated with 0.5% methylcellulose. Consistent with the previous finding (29), the level of apoB-100 in the serum of CP-346086-treated mice was reduced by 79% compared to that of the control group (Fig. 8A). As a result, the inhibitor-treated mouse serum failed to inhibit HCV cell attachment at concentrations of 0.625% and 1.25%, whereas the control mouse serum blocked HCV attachment by 50% and 60%, respectively. At a 2.5% serum concentration, the HCV-inhibitory activity was reduced by about 40% in the MTP inhibitor-treated animals compared to the control animals (Fig. 8B). These results clearly show that treatment of mouse with the MTP inhibitor CP-346086 significantly decreased the level of apoB-100 and therefore the HCV-inhibitory activity in mouse serum.

FIG 8.

FIG 8

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Reduction of the HCV-inhibitory activity of mouse serum upon administration of an MTP inhibitor. Mice were orally administered CP-346086 at 33 mg/kg for 14 days. Mouse blood was collected by bleeding. Serum from 5 animals of each group was pooled. (A) Comparison of apoB levels between control and CP-346086-treated mice. Mouse serum proteins were separated by electrophoresis in a 5% SDS–polyacrylamide gel. apoB was detected by WB using a goat polyclonal antibody. Purified human VLDL, which was used for the experiments described in the legend to Fig. 7, was used as a positive control for apoB detection. (B) Reversal of HCV-inhibitory activity of mouse serum by treatment with CP-346086. Huh-7.5 cells were incubated with HCV in the presence of increasing concentrations (0, 0.625, 1.25, and 2.5%) of mouse serum on ice for 2 h. The vRNA of cell-bound HCV was extracted with TRIzol and quantified using a one-step qRT-PCR method. Average levels of HCV vRNA from three independent experiments are plotted against mouse serum concentrations.

DISCUSSION

The narrow host range of HCV has been the major barrier for the development of small animal models for the study of HCV infection, replication, and pathogenesis in vivo (30). A number of independent groups, including us, have previously shown that the genotype 2a HCV (JFH1) was able to efficiently replicate in various human and murine hepatic and extrahepatic cell types in vitro (31–33), suggesting that HCV RNA replication was not strictly restricted to human hepatocytes. The cell tropism of HCV infection and replication in human hepatocytes was probably determined by expression of a subset of key receptors and coreceptors on the surface of human hepatocytes, including CD81, claudin, occludin, and SR-BI (34). Additionally, microRNA 122 (miR-122) and apolipoprotein E (apoE) were found to be important for efficient HCV replication and virus particle formation, respectively (24, 35). When these key cellular factors were expressed together in nonhepatic cell types, the entire HCV life cycle could be fully recapitulated (36, 37), suggesting that HCV infection is primarily restricted by expression of cell surface receptors. More significantly, transgenic mice expressing key human HCV receptors such as CD81 and occludin became susceptible to HCV infection (18–20). In contrast to its infection in vitro, however, HCV infection and/or replication in the humanized mice was extremely inefficient, as suggested by low viremia and the lack of robust viral protein and RNA detection in the mouse hepatocytes upon HCV inoculation (18–20). The low level of HCV propagation in the humanized animals was likely due to the lack or insufficient expression of cellular factors important for virus replication, the presence of restriction factors that inhibit HCV infection and replication, or both. In the present study, we found that both mouse and human sera contain restriction factors that remarkably inhibited HCV infection in cell culture (Fig. 1 and 2). Specifically, mouse serum blocked HCV attachment to the surface of hepatocytes (Fig. 3). Substantial evidence from our studies demonstrates that VLDL is the major serum restriction factor of HCV infection. Unlike the inactive fractions of mouse serum, all fractions with HCV-inhibitory activity contained apoB-associated lipoproteins (Fig. 4 and 5). Strikingly, purified mouse VLDL completely inhibited HCV infection in vitro (Fig. 6). Likewise, purified human VLDL also significantly suppressed HCV infection (Fig. 7). The blockade of HCV cell attachment by mouse and human sera was likely via a competitive binding of VLDL to the same HCV cell surface attachment receptor(s). We and others have previously shown that HSPGs are important for HCV infection in cell culture (3, 4). We further demonstrated that syndecan-1 serves as the major receptor protein for HCV attachment to the cell surface of hepatocytes (38). Our earlier studies also demonstrated that the cellular protein apoE incorporated onto the viral envelope mediates the binding of HCV to HSPG, which is also a known receptor for VLDL (39). Therefore, VLDL can compete with HCV for binding to the same HSPG receptor and consequently blocks HCV infection. Similar to mouse serum, the HCV-inhibitory activity of human serum was most likely contributed by VLDL since purified human VLDL is also a potent inhibitor of HCV infection (Fig. 7).

It has long been known that HCV is associated with lipoproteins in the plasma of hepatitis C patients (40–45). Lipoproteins apoB, apoC1, and apoE were all detected in the HCV RNA-containing particles, which could also be captured by apoB-, apoC1-, and apoE-specific antibodies (25, 40, 43, 46–48). Delipidation of the low-density HCV particles with detergents resulted in nucleocapsid-like structures (40). Recombinant HCV E1/E2 could bind to apolipoproteins, VLDL, LDL, and HDL (49, 50). Therefore, it was speculated that HCV is physically associated with lipoprotein particles, although other studies suggested that HCV is assembled as lipo-viro-particles (LVPs) (40, 41). It is thought that the lipoproteins associated with HCV virion in vivo might enhance its infectivity. Opposing this view, our findings argue that VLDL in the plasma can actually inhibit HCV infection. Therefore, the plasma VLDL is beneficial to hepatitis C patients by suppressing the transmission and spread of HCV in the liver. This argument is supported by circumstantial evidence derived from a clinical study (51). It was found that a sustained virological response (SVR) to interferon alpha and ribavirin combination therapy was positively correlated with a higher apoB-associated cholesterol level, suggesting that apoB-containing lipoproteins (e.g., VLDL) can compete with HCV for receptor binding and therefore restrict HCV infection and spread in the liver (51). The restriction of HCV infection by VLDL in vivo may also explain the observation that only a small fraction (7% to 20%) of hepatocytes in the liver of hepatitis C patients is infected with HCV. The HCV-infected hepatocytes often appear as clusters, suggesting HCV cell-cell transmission in the liver (52). The cell-cell transmission is an intrinsic property of HCV, as demonstrated by several studies in cell culture (53–55). Whether the HCV cell-cell transmission can escape the VLDL-mediated inhibition warrants further investigation. It will also be interesting to determine whether the viral load inversely correlates with the levels of VLDL in the plasma of patients with hepatitis C.

The findings obtained from this study also suggest that HCV infection in humanized mice can be greatly enhanced by eliminating its restriction factor VLDL from the mouse serum. Circumstantial supporting evidence from our studies suggests that administration of an MTP inhibitor (CP-346086) resulted in a significant reduction of the HCV-inhibitory activity in mouse serum (Fig. 8). Our earlier studies demonstrated that MTP inhibitors could completely block the apoB-containing lipoprotein secretion but did not significantly affect apoE incorporation into HCV particles (25). The findings from our previous studies also demonstrate that apoE but not apoB is critically important for both HCV infection and assembly (24–26). Therefore, elimination of apoB-containing lipoproteins such as VLDL will not affect HCV production but will likely facilitate HCV transmission and spread in the humanized mice. Consequently, the permissiveness of humanized mice to HCV infection can be enhanced by cross-breeding with specific gene knockout mice defective in VLDL production (56). This approach may lead to the development of more robust mouse models of HCV infection and replication for the study of viral pathogenesis and for evaluation of effective HCV vaccines.

ACKNOWLEDGMENTS

This work was supported by NIH/NIAID grants (AI097318 and AI091953) and partially by the Nature Science Foundation of China (NSFC 81130082 and NSFC 81101238). J.N. and M.B.R. were supported in part by NIH grants DK078244, DK082753, and GM098539.

We thank Charlie Rice (Rockefeller University) for providing the Huh-7.5 cell line and NS5A monoclonal antibody 9E10, Takaji Wakita (NIAID, Japan) for the JFH1 replicon cDNA, and Lei Cai (University of Kentucky) for apoE−/− mouse serum.

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