Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles

International audienceObjective: HCV is intimately linked with the liver lipid metabolism, devoted to the efflux of triacylglycerols stored in lipid droplets (LDs) in the form of triacylglycerol-rich very-low-density lipoproteins (VLDLs): (i) the most infectious HCV particles are those of lowest density due to association with triacylglycerol-rich lipoproteins and (ii) HCV-infected patients frequently develop hepatic steatosis (increased triacylglycerol storage). The recent identification of lysophosphatidylcholine acyltransferase 1 (LPCAT1) as an LD phospholipid-remodelling enzyme prompted us to investigate its role in liver lipid metabolism and HCV infectious cycle.Design Huh-7.5.1 cells and primary human hepatocytes (PHHs) were infected with JFH1-HCV. LPCAT1 depletion was achieved by RNA interference. Cells were monitored for LPCAT1 expression, lipid metabolism and HCV production and infectivity. The density of viral particles was assessed by isopycnic ultracentrifugation.Results: Upon HCV infection, both Huh-7.5.1 cells and PHH had decreased levels of LPCAT1 transcript and protein, consistent with transcriptional downregulation. LPCAT1 depletion in either naive or infected Huh-7.5.1 cells resulted in altered lipid metabolism characterised by LD remodelling, increased triacylglycerol storage and increased secretion of VLDL. In infected Huh-7.5.1 cells or PHH, LPCAT1 depletion increased production of the viral particles of lowest density and highest infectivity.Conclusions: We have identified LPCAT1 as a modulator of liver lipid metabolism downregulated by HCV, which appears as a viral strategy to increase the triacylglycerol content and hence infectivity of viral particles. Targeting this metabolic pathway may represent an attractive therapeutic approach to reduce both the viral titre and hepatic steatosis

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

<div><p>In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding <i>per se</i>, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.</p></div

EM visualization of intracellular HCV particles.

<p>Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT) or Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and cell lysates were loaded onto iodixanol gradients. After isopycnic ultracentrifugation, fractions with densities ranging from 1.12 to 1.13 g/mL were pooled, and directly subjected to EM visualization after negative staining (left panels) or delipidated with 0.1% Triton X-100 and subjected to immunogold labeling with mAb against HCV core protein before negative staining and EM visualization (right panels).</p

Titration of extracellular and intracellular infectivity.

<p>Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2), or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, (A) culture supernatants were harvested and probed for extracellular infectivity titers, and (B) cells were lysed by freeze-thaw cycles and probed for intracellular infectivity titers. Mean values of four independent experiments are shown and the standard errors of the means are presented. The threshold of detection of the intracellular infectivity assay is evaluated at 2 ffu per 10⁶ transfected cells.</p

Core protein envelopment analysis by membrane protection assay.

<p>Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and left untreated or treated with 5 μg/mL proteinase K (PK) in the presence or absence of 1% Triton X-100 (Triton). The samples were subjected to (A) western blot analysis with mAb against HCV core protein (a representative western blot is shown) or (B) dot blot for quantification of signal intensities with ImageJ (values were normalized to untreated samples). The mean values and standard errors of four independent experiments are shown. *, P comprised between 0.05 and 0.01; **, P comprised between 0.01 and 0.001; ***, P below 0.001.</p

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection

International audienceBACKGROUND:Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed.METHODS:We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells.RESULTS:We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis.CONCLUSIONS:Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection

Severe COVID-19 is associated with hyperactivation of the alternative complement pathway

International audienceBackground: Severe coronavirus disease 2019 (COVID-19) is characterized by impaired type I interferon activity and a state of hyperinflammation leading to acute respiratory distress syndrome. The complement system has recently emerged as a key player in triggering and maintaining the inflammatory state, but the role of this molecular cascade in severe COVID-19 is still poorly characterized.Objective: We aimed at assessing the contribution of complement pathways at both the protein and transcriptomic levels.Methods: To this end, we systematically assessed the RNA levels of 28 complement genes in the circulating whole blood of patients with COVID-19 and healthy controls, including genes of the alternative pathway, for which data remain scarce.Results: We found differential expression of genes involved in the complement system, yet with various expression patterns: whereas patients displaying moderate disease had elevated expression of classical pathway genes, severe disease was associated with increased lectin and alternative pathway activation, which correlated with inflammation and coagulopathy markers. Additionally, properdin, a pivotal positive regulator of the alternative pathway, showed high RNA expression but was found at low protein concentrations in patients with a severe and critical disease, suggesting its deposition at the sites of complement activation. Notably, low properdin levels were significantly associated with the use of mechanical ventilation (area under the curve = 0.82; P = .002).Conclusion: This study sheds light on the role of the alternative pathway in severe COVID-19 and provides additional rationale for the testing of drugs inhibiting the alternative pathway of the complement system

Production of Infectious Hepatitis C Virus in Primary Cultures of Human Adult Hepatocytes

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