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Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry.

Maillard P, Walic M, Meuleman P, Roohvand F, Huby T, Le Goff W, Leroux-Roels G, Pécheur EI, Budkowska A - PLoS ONE (2011)

Bottom Line: The effect of LPL depended on its enzymatic activity.These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets.HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité Hépacivirus et Immunité Innée, Département de Virologie, Paris, France.

ABSTRACT
A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

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Immunoelectron microscopy of the infection of Huh 7.5 cells in the presence of LPL.Huh7.5 cells were pre-incubated with 1 µg/ml LPL, as described in Materials and Methods. The viral preparation, concentrated by centrifugation through a sucrose cushion, was incubated with cells at 4°C (T0), then transferred to 37°C and incubated for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells were washed, fixed and stained with anti-E2 (AP-33) or anti-core (ACAP-27) monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG. LPL 10 min is a representative view of uninfected cells, pretreated with LPL at 4°C and subsequently for 10 min at 37°C, before immunogold labeling with anti-LPL antibodies and processing for TEM. T0, T10 and T20, show immunogold labeling with antibodies directed against HCV E2 (T0) and core protein (T10 and T20). Asterisks denote the presence of one silver-enhanced gold particle. CCP, clathrin-coated pit; CV, clathrin vesicle; M, mitochondrion.
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pone-0026637-g008: Immunoelectron microscopy of the infection of Huh 7.5 cells in the presence of LPL.Huh7.5 cells were pre-incubated with 1 µg/ml LPL, as described in Materials and Methods. The viral preparation, concentrated by centrifugation through a sucrose cushion, was incubated with cells at 4°C (T0), then transferred to 37°C and incubated for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells were washed, fixed and stained with anti-E2 (AP-33) or anti-core (ACAP-27) monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG. LPL 10 min is a representative view of uninfected cells, pretreated with LPL at 4°C and subsequently for 10 min at 37°C, before immunogold labeling with anti-LPL antibodies and processing for TEM. T0, T10 and T20, show immunogold labeling with antibodies directed against HCV E2 (T0) and core protein (T10 and T20). Asterisks denote the presence of one silver-enhanced gold particle. CCP, clathrin-coated pit; CV, clathrin vesicle; M, mitochondrion.

Mentions: In the presence of LPL, HCV was observed exclusively extracellularly, in close proximity to the plasma membrane, at all time points: T0, T10 and T20 (Figure 8). A very similar distribution was observed with anti-E2 and anti-core antibodies, indicating that, even at longer incubation times, complete HCV particles containing the virus nucleocapsid and envelope were still attached to the plasma membrane. Immunogold labeling of uninfected cells with an antibody against LPL was exclusively confined to the plasma membrane (Figure 8; LPL 10 min), suggesting that the enzyme also remained bound to the plasma membrane, or was degraded inside the cell. Uninfected cells incubated with the buffer used to dissolve LPL alone had a normal morphology and ultrastructure (data not shown).


Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry.

Maillard P, Walic M, Meuleman P, Roohvand F, Huby T, Le Goff W, Leroux-Roels G, Pécheur EI, Budkowska A - PLoS ONE (2011)

Immunoelectron microscopy of the infection of Huh 7.5 cells in the presence of LPL.Huh7.5 cells were pre-incubated with 1 µg/ml LPL, as described in Materials and Methods. The viral preparation, concentrated by centrifugation through a sucrose cushion, was incubated with cells at 4°C (T0), then transferred to 37°C and incubated for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells were washed, fixed and stained with anti-E2 (AP-33) or anti-core (ACAP-27) monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG. LPL 10 min is a representative view of uninfected cells, pretreated with LPL at 4°C and subsequently for 10 min at 37°C, before immunogold labeling with anti-LPL antibodies and processing for TEM. T0, T10 and T20, show immunogold labeling with antibodies directed against HCV E2 (T0) and core protein (T10 and T20). Asterisks denote the presence of one silver-enhanced gold particle. CCP, clathrin-coated pit; CV, clathrin vesicle; M, mitochondrion.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198807&req=5

pone-0026637-g008: Immunoelectron microscopy of the infection of Huh 7.5 cells in the presence of LPL.Huh7.5 cells were pre-incubated with 1 µg/ml LPL, as described in Materials and Methods. The viral preparation, concentrated by centrifugation through a sucrose cushion, was incubated with cells at 4°C (T0), then transferred to 37°C and incubated for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells were washed, fixed and stained with anti-E2 (AP-33) or anti-core (ACAP-27) monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG. LPL 10 min is a representative view of uninfected cells, pretreated with LPL at 4°C and subsequently for 10 min at 37°C, before immunogold labeling with anti-LPL antibodies and processing for TEM. T0, T10 and T20, show immunogold labeling with antibodies directed against HCV E2 (T0) and core protein (T10 and T20). Asterisks denote the presence of one silver-enhanced gold particle. CCP, clathrin-coated pit; CV, clathrin vesicle; M, mitochondrion.
Mentions: In the presence of LPL, HCV was observed exclusively extracellularly, in close proximity to the plasma membrane, at all time points: T0, T10 and T20 (Figure 8). A very similar distribution was observed with anti-E2 and anti-core antibodies, indicating that, even at longer incubation times, complete HCV particles containing the virus nucleocapsid and envelope were still attached to the plasma membrane. Immunogold labeling of uninfected cells with an antibody against LPL was exclusively confined to the plasma membrane (Figure 8; LPL 10 min), suggesting that the enzyme also remained bound to the plasma membrane, or was degraded inside the cell. Uninfected cells incubated with the buffer used to dissolve LPL alone had a normal morphology and ultrastructure (data not shown).

Bottom Line: The effect of LPL depended on its enzymatic activity.These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets.HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité Hépacivirus et Immunité Innée, Département de Virologie, Paris, France.

ABSTRACT
A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

Show MeSH
Related in: MedlinePlus