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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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Analysis of the influence of LPL on VLDL hydrolysis and uptake by Huh 7.5 cells.Huh 7.5 cells were washed with medium without FCS and pre-incubated with LPL on ice. Purified VLDL were then added and, after 30 min, cells were transferred to 37°C and incubated for a further 2 h. The effect of LPL on VLDL uptake was shown by the increase in the amounts of both total triglycerides (TG) associated with cells in the presence of LPL. Cellular levels of TC and TG were also assessed in the presence of LPL and THL or heparin. (A) Hydrolysis of VLDL by LPL increased cellular TG levels due to the intracellular accumulation of fatty acids. This process was inhibited by THL, but was insensitive to the action of heparin, as it did not involve “bridging” mechanisms. (B) LPL increased the levels of TC in the cells in the presence VLDL. The effect of LPL was abolished by adding either THL, an inhibitor of LPL enzyme activity, or heparin, which prevents LPL from associating with proteoglycans. Thus, both catalytic and bridging activities were involved.
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pone-0026637-g004: Analysis of the influence of LPL on VLDL hydrolysis and uptake by Huh 7.5 cells.Huh 7.5 cells were washed with medium without FCS and pre-incubated with LPL on ice. Purified VLDL were then added and, after 30 min, cells were transferred to 37°C and incubated for a further 2 h. The effect of LPL on VLDL uptake was shown by the increase in the amounts of both total triglycerides (TG) associated with cells in the presence of LPL. Cellular levels of TC and TG were also assessed in the presence of LPL and THL or heparin. (A) Hydrolysis of VLDL by LPL increased cellular TG levels due to the intracellular accumulation of fatty acids. This process was inhibited by THL, but was insensitive to the action of heparin, as it did not involve “bridging” mechanisms. (B) LPL increased the levels of TC in the cells in the presence VLDL. The effect of LPL was abolished by adding either THL, an inhibitor of LPL enzyme activity, or heparin, which prevents LPL from associating with proteoglycans. Thus, both catalytic and bridging activities were involved.

Mentions: The effect of LPL on VLDL was already observable after 2 h of incubation, as shown by increases in the levels of both triglycerides (TG) and total cholesterol (TC) associated with the cells in the presence of LPL (Figure 4). We used tetrahydrolipstatin (THL), a specific inhibitor of the catalytic activity of LPL, which blocks the active site of the enzyme [48], to assess the impact of the lipolytic function of LPL on lipoprotein uptake.


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)

Analysis of the influence of LPL on VLDL hydrolysis and uptake by Huh 7.5 cells.Huh 7.5 cells were washed with medium without FCS and pre-incubated with LPL on ice. Purified VLDL were then added and, after 30 min, cells were transferred to 37°C and incubated for a further 2 h. The effect of LPL on VLDL uptake was shown by the increase in the amounts of both total triglycerides (TG) associated with cells in the presence of LPL. Cellular levels of TC and TG were also assessed in the presence of LPL and THL or heparin. (A) Hydrolysis of VLDL by LPL increased cellular TG levels due to the intracellular accumulation of fatty acids. This process was inhibited by THL, but was insensitive to the action of heparin, as it did not involve “bridging” mechanisms. (B) LPL increased the levels of TC in the cells in the presence VLDL. The effect of LPL was abolished by adding either THL, an inhibitor of LPL enzyme activity, or heparin, which prevents LPL from associating with proteoglycans. Thus, both catalytic and bridging activities were involved.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026637-g004: Analysis of the influence of LPL on VLDL hydrolysis and uptake by Huh 7.5 cells.Huh 7.5 cells were washed with medium without FCS and pre-incubated with LPL on ice. Purified VLDL were then added and, after 30 min, cells were transferred to 37°C and incubated for a further 2 h. The effect of LPL on VLDL uptake was shown by the increase in the amounts of both total triglycerides (TG) associated with cells in the presence of LPL. Cellular levels of TC and TG were also assessed in the presence of LPL and THL or heparin. (A) Hydrolysis of VLDL by LPL increased cellular TG levels due to the intracellular accumulation of fatty acids. This process was inhibited by THL, but was insensitive to the action of heparin, as it did not involve “bridging” mechanisms. (B) LPL increased the levels of TC in the cells in the presence VLDL. The effect of LPL was abolished by adding either THL, an inhibitor of LPL enzyme activity, or heparin, which prevents LPL from associating with proteoglycans. Thus, both catalytic and bridging activities were involved.
Mentions: The effect of LPL on VLDL was already observable after 2 h of incubation, as shown by increases in the levels of both triglycerides (TG) and total cholesterol (TC) associated with the cells in the presence of LPL (Figure 4). We used tetrahydrolipstatin (THL), a specific inhibitor of the catalytic activity of LPL, which blocks the active site of the enzyme [48], to assess the impact of the lipolytic function of LPL on lipoprotein uptake.

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