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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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LPL affects HCV attachment and early stages of the virus cell cycle.(A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.
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pone-0026637-g006: LPL affects HCV attachment and early stages of the virus cell cycle.(A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.

Mentions: These data indicated that the changes in the lipid composition of the virus particles induced by catalytically active LPL were only partly responsible for the inhibitory effect of the enzyme on viral infectivity and suggested a predominant role for the second, structure-bridging function of LPL. Indeed, exogenously added LPL enhanced the binding of HCVcc to Huh7.5 cells (at 4°C), as shown by the increase in HCV-RNA associated with cells (Figure 6A). This increase was maximal for a concentration of 1–3 µg/ml LPL. Higher concentrations of LPL did not increase virus binding further and were instead toxic to cells.


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)

LPL affects HCV attachment and early stages of the virus cell cycle.(A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026637-g006: LPL affects HCV attachment and early stages of the virus cell cycle.(A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.
Mentions: These data indicated that the changes in the lipid composition of the virus particles induced by catalytically active LPL were only partly responsible for the inhibitory effect of the enzyme on viral infectivity and suggested a predominant role for the second, structure-bridging function of LPL. Indeed, exogenously added LPL enhanced the binding of HCVcc to Huh7.5 cells (at 4°C), as shown by the increase in HCV-RNA associated with cells (Figure 6A). This increase was maximal for a concentration of 1–3 µg/ml LPL. Higher concentrations of LPL did not increase virus binding further and were instead toxic to cells.

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