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1 Lysophosphatidylcholine acyltransferase 1 () is downregulated by hepatitis C virus: impact on production of lipo-viro-particles Matthieu LEMASSON, Frauke BEILSTEIN, Véronique PENE, Sylvie DEMIGNOT & Arielle R. ROSENBERG AUCUN CONFLIT D'INTERET University Paris Descartes EA 4474 Hepatitis C Virology Paris, FRANCE
2 Circulating HCV particles in the blood of patients Lipo-viro-particles < 1.10 g/ml Enveloped particles 1.12 to 1.21 g/ml Nucleocapsids > 1.25 g/ml Density ApoB Infectivity André et al. J. Virol Nielsen et al. J. Virol Petit et al. Virology 2005 Maillard et al. J. Virol. 2001
3 Model for Model the partition for HCV morphogenesis of lipids in hepatocytes VLDL Pre-VLDL ApoB MTP Luminal lipid droplet Lipo-viro-particle ER Lumen Ribosome Triglycerides + Cholesterol esters HCV core Cytosol Cytosolic lipid droplet Replication complex Membranous web
4 Lysophosphatidylcholine acyltransferase 1 () De novo Land s cycle ER Lumen biosynthesis modified after Shindou et al. J. Lipid Res Acyl-chain symmetry Membrane uniformity PLA2 Acyl-chain asymmetry Membrane diversity Cytosol Phosphocholine Acyl-chain moiety Cytosolic lipid droplet Moessinger et al. J. Biol. Chem. 2011
5 involvement in HCV infectious cycle? expression level Western blot Nucleus ER VLDL-like RT-qPCR Immunofluorescence in situ RNA(+) Membranous web RNA(-) ARN(-) LD HCVcc Huh cells
6 Relative gene expression Relative amount of protein is downregulated upon HCV infection 1.2 expression level HCV core protein Western blot RT-qPCR Immunofluorescence in situ HCV core Lipid droplet Merge 10µm transcript 0.2
7 Impact of knockdown on the lipid metabolism of hepatocytes? shluc sh sh Lipid metabolism Size of lipid droplets (Immunofluorescence in situ) Nucleus ER VLDL-like Intracellular amount of triacylglycerols (Colorimetric assay) Lipid secretion (Metabolic labeling using [1-14 C] oleic acid) LD Huh cells sh vs shluc
8 sh shluc % of total number of lipid droplets knockdown increases the mean size of lipid droplets shluc sh sh Lipid metabolism Size of lipid droplets (Immunofluorescence in situ) Lipid droplet 10µm sh shluc Intracellular amount of triacylglycerols (Colorimetric assay) Lipid secretion (Metabolic labeling using [1-14 C] oleic acid) µm above 1 Size of lipid droplets (µm)
9 Intracellular triacylglycerol amount (% of control) knockdown increases the intracellular amount of triglycerides shluc sh sh Lipid metabolism Size of lipid droplets (Immunofluorescence in situ) Intracellular amount of triacylglycerols (Colorimetric assay) Lipid secretion (Metabolic labeling using [1-14 C] oleic acid) shluc sh
10 Oleic acid incorporated (nmoles / dish) Oleic acid incorporated (nmoles / dish) knockdown increases the secretion of newly synthesized triglycerides shluc sh sh Lipid metabolism Size of lipid droplets (Immunofluorescence in situ) Triacylglycerols Phospholipids Intracellular amount of triacylglycerols (Colorimetric assay) Cells Media Lipid secretion (Metabolic labeling using [1-14 C] oleic acid) sh shluc Phospholipids Triacylglycerols Phospholipids Triacylglycerols
11 Impact of knockdown on HCV infectious cycle? sh Day 6 shluc sh HCV core HCV infectious cycle Replication of HCV genome (Negative strand HCV RNA) Nucleus ER VLDL-like Production of infectious HCV (Focus-formation assay) Production of HCV particles (Standardized viral load assay) RNA(+) RNA(-) ARN(-) Membranous web LD HCVcc Specific infectivity (Ratio = infectious titer ) viral load Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) Huh cells sh vs shluc
12 Fold increase knockdown increases the infectivity of viral particles produced sh Day 6 shluc sh HCV core HCV infectious cycle sh shluc Replication of HCV genome (Negative strand HCV RNA) Production of infectious HCV (Focus-formation assay) Production of HCV particles (Standardized viral load assay) Specific infectivity (Ratio = infectious titer ) viral load Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) Negative strand Infectious titer Viral load Specific infectivity Cells Media Carrière et al. J. Med. Virol. 2007
13 HCV RNA (% of total) knockdown increases the proportion of low-density HCV particles sh Day 6 Infectivity sh shluc Merge Density HCV core shluc sh HCV infectious cycle Replication of HCV genome (Negative strand HCV RNA) Production of infectious HCV (Focus-formation assay) Production of HCV particles (Standardized viral load assay) Specific infectivity (Ratio = infectious titer ) viral load Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) Density (g/ml)
14 involvement in HCV infectious cycle in PHH? Day 0 Day 6 expression level RT-qPCR Nucleus ER VLDL RNA(+) Membranous web RNA(-) ARN(-) LD HCVpc Primary Human Hepatocytes (PHH) Podevin et al. Gastroenterology 2010
15 Relative gene expression Infectious titer (ffu/ml) Relative gene expression is downregulated upon HCV infection in PHH Day 0 Day 6 expression level RT-qPCR R² = y = x Infectious titer (log 10 ffu/ml)
16 Impact of knockdown on HCV infectious cycle in PHH? sigfp si si Day 6 HCV infectious cycle Nucleus ER VLDL Production of infectious HCV (Focus-formation assay) Production of HCV particles (Standardized viral load assay) Specific infectivity infectious titer (Ratio = ) viral load RNA(+) Membranous web Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) RNA(-) ARN(-) LD HCVpc PHH si vs sigfp
17 Fold increase knockdown increases the infectivity of viral particles produced in PHH sigfp si si Day 6 HCV infectious cycle si sigfp Production of infectious HCV (Focus-formation assay) Production of HCV particles (Standardized viral load assay) Specific infectivity infectious titer (Ratio = ) viral load 8 6 Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) 4 2 Infectious titer Viral load Specific infectivity
18 HCV RNA (% of total) knockdown increases the proportion of low-density HCV particles in PHH sigfp si si Day 6 Density HCV infectious cycle Production of infectious HCV (Focus-formation assay) Infectivity si sigfp Merge Production of HCV particles (Standardized viral load assay) Specific infectivity infectious titer (Ratio = ) viral load Density of HCV particles (Isopycnic ultracentrifugation in iodixanol gradient) Density (g/ml)
19 Model for downregulation as a viral strategy to increase the production of lipo-viro-particles VLDL VLDL Lipo-viro-particle LD Non infected human hepatocytes ER Lumen ER Lumen Cytosol Cytosol LD HCV-infected human hepatocytes
20 Acknowledgments Véronique PENE Philippe PODEVIN Jean-François MERITET Yves BENHAMOU Frauke BEILSTEIN Arielle R. ROSENBERG Sylvie DEMIGNOT EA 4474 Hepatitis C Virology University Paris Descartes Paris, France UMRS 872 Centre de Recherche des Cordeliers Paris, France This work was supported by the ANRS (France REcherche Nord & sud Sida-hiv Hépatites : FRENSH)
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