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Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

Dreux M, Dao Thi VL, Fresquet J, Guérin M, Julia Z, Verney G, Durantel D, Zoulim F, Lavillette D, Cosset FL, Bartosch B - PLoS Pathog. (2009)

Bottom Line: This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI.Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement.Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, UCB-Lyon1, IFR128, INSERM, U758, Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

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HCVcc entry in SK-Hep1-CLDN1 cells expressing SR-BI mutants.The SR-BI mutants tested were the SR-BI/CD36 chimera and the SES mutants,                            altering SR-BI intracellular domain, the Q402R/E418R, N173Q, and                            G420H-G424H mutants, altering SR-BI ectodomain. (A) Effect of SR-BI                            mutations on infectivity of HCVcc produced under standard conditions and                            assessed by measuring intracellular HCV RNA level at 72 hr                            post-infection (see Figure                                2A). The results of infectivity (mean±SD;                            n = 3) are expressed relative to HCVcc                            infection of wt SR-BI–expressing SK-Hep1-CLDN1 cells, which                            was set to 100. (B) Results of HCVcc infection-enhancement induced by                            HDL (0.6 µg/ml cholesterol-HDL) on wt                            SR-BI–expressing SK-Hep1-CLDN1 cells, expressed as ratios                            relative to infection in the absence of HDL of the same cells set to 100                            (mean±SD; n = 3). Similar                            experiments in BRL3A-CD81-CLDN1 cells expressing these SR-BI mutants                            were performed (data not shown) and were consistent with the results                            obtained in SK-Hep1-CLDN1 cells.
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ppat-1000310-g004: HCVcc entry in SK-Hep1-CLDN1 cells expressing SR-BI mutants.The SR-BI mutants tested were the SR-BI/CD36 chimera and the SES mutants, altering SR-BI intracellular domain, the Q402R/E418R, N173Q, and G420H-G424H mutants, altering SR-BI ectodomain. (A) Effect of SR-BI mutations on infectivity of HCVcc produced under standard conditions and assessed by measuring intracellular HCV RNA level at 72 hr post-infection (see Figure 2A). The results of infectivity (mean±SD; n = 3) are expressed relative to HCVcc infection of wt SR-BI–expressing SK-Hep1-CLDN1 cells, which was set to 100. (B) Results of HCVcc infection-enhancement induced by HDL (0.6 µg/ml cholesterol-HDL) on wt SR-BI–expressing SK-Hep1-CLDN1 cells, expressed as ratios relative to infection in the absence of HDL of the same cells set to 100 (mean±SD; n = 3). Similar experiments in BRL3A-CD81-CLDN1 cells expressing these SR-BI mutants were performed (data not shown) and were consistent with the results obtained in SK-Hep1-CLDN1 cells.

Mentions: While these results were obtained in a rat hepato-carcinoma background, they were largely confirmed in the human background of SK-Hep1-CLDN1 cells expressing the different SR-BI mutants (Figure S4). Furthermore, we tested a subset of these SR-BI mutants in HCVcc infection assays. While CD36 expression in SK-Hep1-CLDN1 cells did not induce infection of HCVcc, the SR-BI/CD36 chimera and the SES mutant were functional, but, for the latter, infection was reduced in comparison to wt SR-BI (Figure 4), in line with results obtained with HCVpp infection assays. Finally, we found that HDL stimulated HCVpp entry (Figure 3 and Figure S4) and HCVcc (Figure 4) at similar levels for SR-BI cytoplasmic tail mutants as compared to wt SR-BI. Altogether, these results indicated that the C-terminal cytoplasmic tail of SR-BI modulates the basal HCV entry process, but seems not to influence HDL-mediated infection-enhancement. This latter observation is consistent with the fact that the SR-BI C-terminal mutants mediated efficient lipid transfer (Figure 3D).


Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

Dreux M, Dao Thi VL, Fresquet J, Guérin M, Julia Z, Verney G, Durantel D, Zoulim F, Lavillette D, Cosset FL, Bartosch B - PLoS Pathog. (2009)

HCVcc entry in SK-Hep1-CLDN1 cells expressing SR-BI mutants.The SR-BI mutants tested were the SR-BI/CD36 chimera and the SES mutants,                            altering SR-BI intracellular domain, the Q402R/E418R, N173Q, and                            G420H-G424H mutants, altering SR-BI ectodomain. (A) Effect of SR-BI                            mutations on infectivity of HCVcc produced under standard conditions and                            assessed by measuring intracellular HCV RNA level at 72 hr                            post-infection (see Figure                                2A). The results of infectivity (mean±SD;                            n = 3) are expressed relative to HCVcc                            infection of wt SR-BI–expressing SK-Hep1-CLDN1 cells, which                            was set to 100. (B) Results of HCVcc infection-enhancement induced by                            HDL (0.6 µg/ml cholesterol-HDL) on wt                            SR-BI–expressing SK-Hep1-CLDN1 cells, expressed as ratios                            relative to infection in the absence of HDL of the same cells set to 100                            (mean±SD; n = 3). Similar                            experiments in BRL3A-CD81-CLDN1 cells expressing these SR-BI mutants                            were performed (data not shown) and were consistent with the results                            obtained in SK-Hep1-CLDN1 cells.
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Related In: Results  -  Collection

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ppat-1000310-g004: HCVcc entry in SK-Hep1-CLDN1 cells expressing SR-BI mutants.The SR-BI mutants tested were the SR-BI/CD36 chimera and the SES mutants, altering SR-BI intracellular domain, the Q402R/E418R, N173Q, and G420H-G424H mutants, altering SR-BI ectodomain. (A) Effect of SR-BI mutations on infectivity of HCVcc produced under standard conditions and assessed by measuring intracellular HCV RNA level at 72 hr post-infection (see Figure 2A). The results of infectivity (mean±SD; n = 3) are expressed relative to HCVcc infection of wt SR-BI–expressing SK-Hep1-CLDN1 cells, which was set to 100. (B) Results of HCVcc infection-enhancement induced by HDL (0.6 µg/ml cholesterol-HDL) on wt SR-BI–expressing SK-Hep1-CLDN1 cells, expressed as ratios relative to infection in the absence of HDL of the same cells set to 100 (mean±SD; n = 3). Similar experiments in BRL3A-CD81-CLDN1 cells expressing these SR-BI mutants were performed (data not shown) and were consistent with the results obtained in SK-Hep1-CLDN1 cells.
Mentions: While these results were obtained in a rat hepato-carcinoma background, they were largely confirmed in the human background of SK-Hep1-CLDN1 cells expressing the different SR-BI mutants (Figure S4). Furthermore, we tested a subset of these SR-BI mutants in HCVcc infection assays. While CD36 expression in SK-Hep1-CLDN1 cells did not induce infection of HCVcc, the SR-BI/CD36 chimera and the SES mutant were functional, but, for the latter, infection was reduced in comparison to wt SR-BI (Figure 4), in line with results obtained with HCVpp infection assays. Finally, we found that HDL stimulated HCVpp entry (Figure 3 and Figure S4) and HCVcc (Figure 4) at similar levels for SR-BI cytoplasmic tail mutants as compared to wt SR-BI. Altogether, these results indicated that the C-terminal cytoplasmic tail of SR-BI modulates the basal HCV entry process, but seems not to influence HDL-mediated infection-enhancement. This latter observation is consistent with the fact that the SR-BI C-terminal mutants mediated efficient lipid transfer (Figure 3D).

Bottom Line: This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI.Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement.Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, UCB-Lyon1, IFR128, INSERM, U758, Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

Show MeSH
Related in: MedlinePlus