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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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Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo.The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods. Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.
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pone-0026637-g002: Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo.The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods. Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.

Mentions: For the JFH-1 strain, HCV-RNA was detected at various concentrations in most fractions of the gradient with a peak at a density of 1.066 g/ml. HCV core antigen was found mostly in two peaks, delineating two viral populations (Figure 2A). The first of these populations banded in the density range of 1.022–1.036 g/ml and colocalized with most of the Apo B and cholesterol (detected in the density range 1.017–1.04 g/ml) (Figure 2B). The second viral peak, containing higher levels of HCV core antigen, partly coincided with HCV RNA and was recovered at a mean density of 1.086 g/ml. ApoB and cholesterol were also found at this density, but in lower concentrations than in the lower-density virus population. A similar distribution of the viral RNA was obtained for the J6/JFH-1strain (Figure 2E). For both virus strains, fractions corresponding to the main HCV RNA peak were of relatively low infectivity (Figure 2F). Thus, there was probably a large excess of RNA over infectious virus particles in this peak. Indeed, the high density population was found to be about 8 to 10 times less infectious than the low-density viral peak. These findings confirm previous observations of an inverse relationship between virus density and infectivity [37], [38], [46], [47].


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)

Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo.The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods. Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026637-g002: Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo.The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods. Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.
Mentions: For the JFH-1 strain, HCV-RNA was detected at various concentrations in most fractions of the gradient with a peak at a density of 1.066 g/ml. HCV core antigen was found mostly in two peaks, delineating two viral populations (Figure 2A). The first of these populations banded in the density range of 1.022–1.036 g/ml and colocalized with most of the Apo B and cholesterol (detected in the density range 1.017–1.04 g/ml) (Figure 2B). The second viral peak, containing higher levels of HCV core antigen, partly coincided with HCV RNA and was recovered at a mean density of 1.086 g/ml. ApoB and cholesterol were also found at this density, but in lower concentrations than in the lower-density virus population. A similar distribution of the viral RNA was obtained for the J6/JFH-1strain (Figure 2E). For both virus strains, fractions corresponding to the main HCV RNA peak were of relatively low infectivity (Figure 2F). Thus, there was probably a large excess of RNA over infectious virus particles in this peak. Indeed, the high density population was found to be about 8 to 10 times less infectious than the low-density viral peak. These findings confirm previous observations of an inverse relationship between virus density and infectivity [37], [38], [46], [47].

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