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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 with HCV in the absence of LPL.The viral preparation (JFH-1) was concentrated by centrifugation through a sucrose cushion and incubated with Huh7.5 cells at 4°C (T0), before transfer to 37°C and incubation for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells collected at all these time points were washed, fixed and stained with monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG (see Materials and Methods section for details). T0 and T5, immunogold labeling of HCV E2 envelope glycoprotein with monoclonal AP-33 antibody; T10 and T20, immunogold labeling of HCV core protein with monoclonal ACAP-27 antibody. Asterisks indicate the presence of one silver-enhanced gold particle. ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondrion; Nu, nucleus.
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pone-0026637-g007: Immunoelectron microscopy of the infection of Huh 7.5 cells with HCV in the absence of LPL.The viral preparation (JFH-1) was concentrated by centrifugation through a sucrose cushion and incubated with Huh7.5 cells at 4°C (T0), before transfer to 37°C and incubation for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells collected at all these time points were washed, fixed and stained with monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG (see Materials and Methods section for details). T0 and T5, immunogold labeling of HCV E2 envelope glycoprotein with monoclonal AP-33 antibody; T10 and T20, immunogold labeling of HCV core protein with monoclonal ACAP-27 antibody. Asterisks indicate the presence of one silver-enhanced gold particle. ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondrion; Nu, nucleus.

Mentions: In the absence of LPL, at the initial attachment phase, HCV was visible only on the outside of the cell, mostly in contact with the hepatocyte plasma membrane (Figure 7, T0). HCV appeared in intracellular vesicles after attachment of the virus at 4°C, followed by 5 min of incubation at 37°C (T5). After longer periods of incubation (10–20 min) at 37°C, HCV progressed further into the cell and was seen in vesicles localized in the cell center (T10), close to lipid droplets, and eventually close to the nucleus (T20). We chose to label both the HCV E2 and HCV core proteins, reasoning that the signal for HCV E2 might be lost at later time points, during viral internalization. However, we noted that the structures labeled with antibodies against E2 or core antibodies were identical, at all time points. The identical distributions of core and E2 suggested that our electron microscopy analyses followed the internalization of complete virus particles.


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 with HCV in the absence of LPL.The viral preparation (JFH-1) was concentrated by centrifugation through a sucrose cushion and incubated with Huh7.5 cells at 4°C (T0), before transfer to 37°C and incubation for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells collected at all these time points were washed, fixed and stained with monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG (see Materials and Methods section for details). T0 and T5, immunogold labeling of HCV E2 envelope glycoprotein with monoclonal AP-33 antibody; T10 and T20, immunogold labeling of HCV core protein with monoclonal ACAP-27 antibody. Asterisks indicate the presence of one silver-enhanced gold particle. ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondrion; Nu, nucleus.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026637-g007: Immunoelectron microscopy of the infection of Huh 7.5 cells with HCV in the absence of LPL.The viral preparation (JFH-1) was concentrated by centrifugation through a sucrose cushion and incubated with Huh7.5 cells at 4°C (T0), before transfer to 37°C and incubation for a further 5 (T5), 10 (T10), 15 (T15) or 20 (T20) min. Cells collected at all these time points were washed, fixed and stained with monoclonal antibodies, followed by secondary, colloidal gold-labeled anti-mouse IgG (see Materials and Methods section for details). T0 and T5, immunogold labeling of HCV E2 envelope glycoprotein with monoclonal AP-33 antibody; T10 and T20, immunogold labeling of HCV core protein with monoclonal ACAP-27 antibody. Asterisks indicate the presence of one silver-enhanced gold particle. ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondrion; Nu, nucleus.
Mentions: In the absence of LPL, at the initial attachment phase, HCV was visible only on the outside of the cell, mostly in contact with the hepatocyte plasma membrane (Figure 7, T0). HCV appeared in intracellular vesicles after attachment of the virus at 4°C, followed by 5 min of incubation at 37°C (T5). After longer periods of incubation (10–20 min) at 37°C, HCV progressed further into the cell and was seen in vesicles localized in the cell center (T10), close to lipid droplets, and eventually close to the nucleus (T20). We chose to label both the HCV E2 and HCV core proteins, reasoning that the signal for HCV E2 might be lost at later time points, during viral internalization. However, we noted that the structures labeled with antibodies against E2 or core antibodies were identical, at all time points. The identical distributions of core and E2 suggested that our electron microscopy analyses followed the internalization of complete virus particles.

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