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The interaction of natural hepatitis C virus with human scavenger receptor SR-BI/Cla1 is mediated by ApoB-containing lipoproteins

Patrick Maillard^*, Thierry Huby, Ursula Andréo^*, Martine Moreau, John Chapman and Agata Budkowska^*,1

^* Hepacivirus Unit, Pasteur Institute, Paris, France; and
INSERM Unit 551, UPMC Paris 6, Dyslipoproteinemia and Atherosclerosis Research Unit, Paris, France

¹Correspondence: Pasteur Institute, Hepacivirus Unit, 25/28 rue du Dr Roux, Paris 75724, France. E-Mail: abudkow{at}pasteur.fr

SPECIFIC AIMS

The molecular mechanisms involved in the uptake of HCV from serum into target cells remain unclear. The human "scavenger receptor" SR-BI/Cla1 was recently identified as a new HCV receptor candidate. SR-BI/Cla1 binds E2 envelope glycoprotein, and mediates the entry of HCV pseudoparticles harboring E1E2 HCV envelope proteins on their surface (HCVpp) into hepatoma cells in the presence of CD-81 as a co-receptor. The capacity of SR-BI/Cla1 to recognize serum HCV and HCV produced in a new in vitro cell culture system (HCV cc) has not yet been investigated, and the putative role of this receptor in HCV infection remains to be defined.

We evaluated whether 1) HCV circulating in the sera of infected individuals interacts with SR-BI/Cla1 expressed on the cell surface, 2) SR-BI/Cla1 mediates the uptake of virus into the cell in the absence of CD-81; and 3) the mechanism of interaction of natural HCV with SR-BI/Cla1 is dependent on HCV envelope proteins or is mediated by virus-associated lipoproteins.

PRINCIPAL FINDINGS

1. Natural HCV interacts with SR-BI/Cla1
Human hepatocytes, the main target of HCV infection, express SR-BI and other candidate receptors, such as CD-81 and LDL-R, and possibly other factors that could mediate viral entry. We developed an experimental in vitro system based on the stable transfection of the CHO cell line with a cDNA encoding human SR-BI/Cla1 in order to assess the capacity of HCV from sera of infected individuals to interact specifically with this receptor. We demonstrated that SR-BI/Cla1 expressed on transfected CHO cells was fully functional by analyzing 1) the binding and internalization of AcLDL; 2)the cellular efflux of free cholesterol mediated by SR-BI/Cla1 to specific ligands (notably, HDL, LDL, and VLDL); and 3) the capacity of SR-BI to bind HCV E2 protein. The capacity of HCV to interact with SR-BI/Cla1 was assessed by incubating serum samples from HCV carriers (genotype 1a or 1b) with transfected or parental CHO cells (as a control) and determining the amount of HCV RNA associated with cells by quantitative real-time RT-PCR, the only available method for detecting HCV in the absence of viral replication.

Significantly larger amounts of HCV RNA were found associated with transfected cell clones as compared with parental CHO cells for 9 of the 10 serum samples analyzed (P<0.05 by Mann-Whitney U test). Viral load in these sera ranged from 22,104 to 3,918,280 IU/mL, as determined by b-DNA assay. The amount of HCV RNA bound by transfected cells and viral load in serum were not directly correlated.

The key role of SR-BI/Cla1 in this process was confirmed by inhibition of virus/cell interaction with antibody directed against the extracellular domain of the receptor (Fig. 1 ). This antibody also interfered with SR-BI/Cla1-mediated free cholesterol efflux to HDL, LDL, and VLDL, a specific function of SR-BI dependent on lipoprotein binding to the receptor.

View larger version (14K): [in this window] [in a new window]   Figure 1. Anti-SR-BI antibodies inhibit the interaction of HCV with SR-BI/Cla1. Transfected CHO K7 and CHO K16 or control CHO cells were incubated for 30 min at 37°C with serial dilutions of rabbit anti-human SR-BI antibodies directed against the extracellular loop of the receptor. HCV-positive serum was then added and the cells were incubated for 2 h at 37°C. Cells were washed, total RNA was extracted and HCV-RNA quantified by RT-PCR. The amount of HCV RNA is expressed in IU.

2. Lysosome acidification-dependent cellular trafficking is involved in HCV uptake via SR-BI/Cla1
The quantity of HCV-RNA associated with transfected cells at 37°C was markedly superior to that associated with cells at 4°C or 14°C, suggesting that SR-BI/Cla1 mediated trafficking of HCV into the cell by energy-dependent mechanisms. Treatment of cells with suramin after inoculation revealed that the majority of HCV RNA (80%) remained associated with transfected cells, consistent with their internalization. Moreover, lysosomotropic agents such as NH₄Cl and amantadine, which raise luminal pH, significantly decreased the amount of HCV-RNA associated with transfected cells in a dose-dependent manner. Thus, the acidification of endosomes is critical to the uptake of natural HCV into cells via SR-BI/Cla1.

3. Interaction of HCV with SR-BI/Cla1 is not mediated by the E2 envelope glycoprotein
We evaluated whether the interaction between HCV and SR-BI/Cla1 occurred directly, via the HCV envelope, or indirectly, via plasma lipoproteins associated to the virus. We attempted to inhibit the HCV/SR-BI interaction using well defined anti-envelope antibodies. Human IgG (HCV-IG) prepared from patients’ sera and containing anti-E1 and anti-E2 antibodies (which neutralized the infection of liver cells with HCVpp in vitro) did not inhibit the interaction of natural HCV with SR-BI/Cla1-expressing cells (Fig. 2 A). Mouse polyclonal, broadly reactive antibodies directed to the hypervariable domain of the HCV E2 envelope glycoprotein (HVR1), the region able to bind the receptor directly, had no effect on virus/cell interaction (Fig. 2B ). Thus, natural HCV did not recognize the receptor via the HVR1 domain or other regions of the HCV E2 glycoprotein.

View larger version (28K): [in this window] [in a new window]   Figure 2. Interaction of HCV with SR-BI/Cla1 is mediated by apo-B-containing lipoproteins. Anti-envelope antibodies do not inhibit interaction of HCV with SR-BI/Cla1. HCV-positive serum samples were incubated with increasing concentrations of HCV-IG or with normal human IgG as a control (A) or with polyclonal anti-HVR1 antibodies or pre-immune mouse (pre-imm) serum as a control for 1 h at 37°C (B) before incubation with CHO-K16 cells for 2 h at 37°C. CHO-K16 cells incubated without antibodies are shown (w/o). Interaction of HCV with SR-BI/Cla1 is inhibited by plasma lipoproteins. C) Purified plasma lipoproteins (VLDL, LDL, or HDL at specified concentrations) were incubated with CHO-K16 cells for 15 min at 37°C and then with HCV-positive serum for 2 h at 37°C. D) Inhibition of HCV/SR-BI interaction by anti-ßlipoprotein antibodies. Patients’ sera were incubated with goat polyclonal antibodies against human ßlipoproteins for 1h at 37°C and then for 2 h at 37°C with CHO-K16 and CH-K7 cells. HCV associated with the cells was quantified by RT-PCR and expressed in IU. HCV RNA bound to control CHO cells is shown (open circle), together with the mean values and standard deviation of six independent analyses.

4. Apo-B-containing lipoproteins mediate virus uptake via SR-BI
We further investigated whether natural SR-BI/Cla1 ligands (HDL, LDL, and VLDL) could compete with the virus for binding to this receptor. Transfected and nontransfected cells were incubated with lipoproteins, then with HCV-positive sera. VLDL inhibited HCV/SR-BI interaction efficiently (by up to 80%), whereas LDL had a much less pronounced effect (Fig. 2C ). HDL had no effect on HCV/SR-BI/Cla1 interaction. HCV/SR-BI interaction was also inhibited in a dose-dependent manner by anti-ß lipoprotein (VLDL and LDL) antibodies (Fig. 2D ). Thus, HCV interacted with SR-BI/Cla1 via apo-B-containing lipoproteins, mainly VLDL.

5. Natural HCV binds to hepatic cells via Apo-B-containing lipoproteins
We validated our findings in CHO cells expressing SR-BI/Cla1 using human hepatoma cell lines HepG2 and Huh7. These cells share similar properties with human hepatocytes and are permissive for HCV replication; however, only the Huh7 line supports infection with HCVpp, which requires CD-81 as a co-receptor. Both cell lines express SR-BI and LDL receptors.

HepG2 and Huh7 cells efficiently bound HCV. Prior incubation of cells with anti-SR-BI antibodies decreased the amount of HCV RNA associated with cells by 40%, showing that the interaction of natural HCV is mediated at least in part by SR-BI. As in the CHO-SR-BI model, HCV-IgG did not inhibit the interaction of natural virus with hepatic cells. VLDL strongly inhibited the interaction of HCV, whereas HDL and LDL had little or no effect. Finally, the preincubation of HCV-positive sera with anti-ß lipoprotein antibodies inhibited HCV/cell interaction in a dose-dependent manner.

CONCLUSIONS AND SIGNIFICANCE

We provide the first direct demonstration that human SR-BI/Cla1 mediates the binding to cells of natural HCV from the serum of infected individuals and its subsequent cellular uptake in the absence of another HCV receptor candidate, CD-81. We demonstrate that lysosome acidification-dependent cellular trafficking was involved in HCV uptake via SR-BI/Cla1. The key role of SR-BI/Cla1 in this process was confirmed by inhibition of virus/cell interaction with an antibody directed against the extracellular domain of the receptor. In contrast to earlier observations with HCVpp as a model of the natural virus, the interaction between serum HCV and SR-BI/Cla1 was not mediated by the HVR1 domain or other regions of E2 envelope glycoprotein. Rather, VLDL either incorporated or associated to the virus particles played a critical role in the primary interaction of authentic virus with SR-BI at the cell surface (Fig. 3 ). All of the key findings obtained in our experimental cellular system (CHO-SR-BI/Cla1) correlated well with data obtained using human HepG2 and Huh7 cell lines. The primary interaction of serum HCV with hepatic cells was not inhibited by either anti-HVR1 or anti-E2 antibodies, which neutralized the infection of Huh7 cells with HCVpp and HCVcc. In contrast, VLDL and anti-ßlipoprotein antibodies efficiently inhibited HCV-cell interaction.

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram. HCV associated with VLDL (1) binds to SR-BI/Cla1 via VLDL (2). Anti-SR-BI antibodies (3), VLDL (4) and anti-ß lipoprotein antibodies (5) inhibit the interaction of HCV with SR-BI/Cla1, whereas anti-E2 and anti-HVR1 antibodies do not (6).

Our findings suggest the existence of an alternative mechanism for the interaction of natural virus with human hepatocytes that is dependent on VLDL and SR-BI. This mechanism is distinct from that mediated by CD-81 and operative for both HCVpp and HCVcc. In contrast to HCVpp and HCVcc, HCV in the serum of infected individuals is highly heterogeneous in nature and most of the HCV in patients’ sera is associated with lipoproteins (mainly VLDL). Only very low density fractions of serum HCV have been shown to transmit infection to chimpanzees and to infect different cell types in vitro via the LDL-R. It is conceivable that primary interaction of natural virus with its target cell might be mediated by lipoproteins associated to virus particles (VLDL and LDL). Nonetheless, we cannot exclude the possibility that E1 and E2 envelope proteins may play a role in later stages of HCV uptake or, alternatively, in HCV morphogenesis and exit from the cell in which CD-81 may act as an "exit receptor" for HCV.

Our data are consistent with the relative inefficiency of anti-envelope antibodies in HCV infection, and suggest that lipoproteins might promote cellular uptake of the virus in the presence of potentially neutralizing antibodies. Free lipoproteins, nonassociated with HCV particles, may compete with lipoprotein-associated HCV for binding to lipoprotein receptors (SR-BI/Cla1 and LDL-R), thereby facilitating HCV clearance; as such, these observations could have key implications for future therapeutic approaches targeted to inhibit HCV infection.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4728fje;

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