Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Abstract

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8,40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (Ͻ50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18,27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a singlestranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5Ј and 3Ј ends. The 5Ј and 3Ј UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor...

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

“…The association appears to occur during assembly, because HCV formation and secretion depend on components of the very low density lipoprotein (VLDL) assembly and secretion pathways, including apolipoprotein E (apoE) and microsomal triglyceride transfer protein (19 -23). Whether apolipoprotein B (apoB) is also required for HCV assembly is discussed controversially (19,21,22,24). ApoB-containing lipo-viro-particles have been identified in patients (16).…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…It seems that HCV can hijack this function for its own assembly as it circulates in patient serum in association with lipoproteins [22,23]. Moreover, host factors crucial for production of VLDL like apolipoprotein E, apolipoprotein B and microsomal triglyceride transfer protein have been found to be important for production and release of infectious HCV particles [24][25][26][27][28][29]. The association of HCV with lipoproteins influences cell entry with the involvement of several lipoprotein (LDL receptor) [30,31] and lipid receptors (Scavenger receptor class B type I, SR-BI; Niemann-Pick C1-like 1 cholesterol uptake receptor, NPC1L1) [32,33].…”

“…Anti-E1/E2 Abs [9,11,62,148], patient sera [9], soluble E2 [46], lectins [150,151], anti-ApoE antibodies [29], apoE peptides [149] Abs, Erlotinib, Lapatinib, Gefitinib [141] EGF, TGF-α [141] HDL [138] ApoCI [139] BLTs [138,140] Abs, Dasatinib [141] Arbidol [144] Abs [109,137], ITX 5061 [134], natural ligands [135,136] Abs, soluble CD81 [9,11] Abs [142] Cldn1 peptide [143] Heparin, GAG nzymatic digestion [69,132] Abs, natural ligands, soluble LDL receptor [30,133] GAGs LDLR CD81 SR-BI Cldn1 Ocln…”

“…Anti-E1/E2 Abs [9,11,62,148], patient sera [9], soluble E2 [46], lectins [150,151], anti-ApoE antibodies [29], apoE peptides [149] Abs, Erlotinib, Lapatinib, Gefitinib [141] EGF, TGF-α [141] HDL [138] ApoCI [139] BLTs [138,140] Abs, Dasatinib [141] Arbidol [144] …”

Hepatitis C virus and proprotein convertase subtilisin/kexin type 9: a detrimental interaction to increase viral infectivity and disrupt lipid metabolism

“…HCV production was reduced also by blocking VLDL assembly through knocking‐down or inhibition of MTP 45, 46. However, these observations were not always confirmed 47, 48, probably because of differences in VLDL synthesis capacity between different cell culture systems. It has been demonstrated that apoE interaction with HCV NS5A is crucial for the viral assembly process 49.…”