Apolipoprotein E Interacts With Hepatitis C Virus Nonstructural Protein 5A and Determines Assembly of Infectious Particles

Abstract

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide.Restriction of HCV infection to human hepatocytes suggests that liver-specific host factors play a role in the viral life cycle. Using a yeast-two-hybrid system, we identified apolipoprotein E (apoE) as a liver-derived host factor specifically interacting with HCV nonstructural protein 5A (NS5A) but not with other viral proteins. The relevance of apoE-NS5A interaction for viral infection was confirmed by co-immunoprecipitation and co-localization studies of apoE and NS5A in an infectious HCV cell culture model system. Silencing apoE expression resulted in marked inhibition of infectious particle production without affecting viral entry and replication. Analysis of particle production in liver-derived cells with silenced apoE expression showed impairment of infectious particle assembly and release. The functional relevance of the apoE-NS5A interaction for production of viral particles was supported by loss or decrease of apoE-NS5A binding in assembly-defective viral mutants. H epatitis C virus (HCV) is a major cause of liver disease, including liver cirrhosis and hepatocellular carcinoma. 1 Current treatment by interferon-alpha and ribavirin is limited by resistance, toxicity, and high costs. 1,2 Novel treatment approaches are therefore urgently needed. HCV is an enveloped single-stranded RNA virus of positive polarity that is a member of the genus Hepacivirus within the family Flaviviridae. 3,4 The HCV RNA genome encodes a unique polyprotein of approximately 3000 amino acids and is flanked at its 5Ј and 3Ј ends by two highly conserved untranslated regions involved in the transla- Conclusion: These results suggest that recruitment of apoE by NS5A is important for viral assembly and release of infectious viral particles. These findings have important implications

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

“…We first generated VL:JFH1 HCVcc by cotransfecting HCV RNA alone, with a scrambled nucleotide control (siCtrl), or with siRNA that targets apoE expression (siApoE). Similar to the range reported previously, 24,27,28 silencing of apoE expression in Huh7.5.1 producer cells resulted in a marked (50%À70%) and significant decrease (P < .001) in infectivity of viruses relative to viruses with unchanged apoE expression. Exposure of VL:JFH1 HCVcc produced from apoE-depleted cells to multiple patient sera with chronic HCV infection modified these HCVcc to be highly sensitive to nAbs ( Figure 1A).…”

“…HCV RNA from immunoprecipitated viruses was isolated using RNeasy extraction (Qiagen, Valencia, CA) and quantified using reverse transcription quantitative PCR as described previously. 13,24 E2-apoE co-immunoprecipitation using lysates of Huh7/ LunetCD81H cells stably expressing ApoE WT or ApoE HA and transfected with VL:JFH1 or the 447 mutant were performed as described elsewhere. 26 Immunoprecipitation of apoE from Huh7.5.1 cells was performed as described previously.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…RNAi-mediated knockdown of ApoB and ApoE as well as pharmacological inhibition of microsomal triglyceride transfer protein (MTP), an enzyme crucial for ApoB maturation and VLDL secretion, have been proven effective in inhibiting HCV production in Huh7 cells, suggesting that secretion of HCV relies on the VLDL pathway. 83,[158][159][160][161][162] These results raise the question of how VLDL is formed and what the link is to HCV assembly. In hepatocytes, free fatty acids are converted to triacylglycerols (TGs) and incorporated into three different structures: the cytoplasmic lipid droplets (here simply referred to as lipid droplets; LDs); ApoB-containing precursors of VLDL (pre-VLDL particles or VLDL2); and luminal ApoBfree lipid droplets (luLDs).…”

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

“…Given the interaction of apoE with NS5A (22,23,56) and the important role of cyclophilin A (CypA) for HCV replication and assembly (57-59) we also probed the density fractions for the presence of these two proteins. In fact, both proteins were detected in these density gradient fractions, with the NS5A peak coinciding with the E2 peak, whereas CypA amounts were highest in fractions containing the highest core protein amounts.…”