Human Apolipoprotein E Is Required for Infectivity and Production of Hepatitis C Virus in Cell Culture

Abstract

Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNAcontaining particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE-and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.Hepatitis C virus (HCV) is a major cause of liver diseases, affecting approximately 170 million people worldwide (59). Most (ϳ85%) acutely HCV-infected individuals become chronic carriers that can develop cirrhosis and hepatocellular carcinoma (50). HCV is an enveloped RNA virus with a singlestrand and positive-sense RNA genome and is classified as Hepacivirus in the Flaviviridae family (47). The genomic RNA consists of a long open reading frame and relatively short untranslated regions (UTR) at the 5Ј and 3Ј ends (11,32,36,46,53). The 5Ј and 3Ј UTR contain cis-acting RNA elements important for HCV polyprotein translation and RNA replication (16-18, 38, 39, 61, 62). The translation of HCV polyprotein is mediated by the internal ribosomal entry site within the 5Ј UTR (46, 58). Upon translation, the HCV polyprotein is cleaved by cellular peptidases and viral proteases into different viral proteins in the order of C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (36, 38). A number of studies demonstrated that the NS3 to NS5B proteins are sufficient for HCV RNA replication (4, 6, 37), which occurs in the membranebound replication complex consisting of HCV RNA and proteins as well as cellular proteins (13,14,42,57). The core and NS5B coding regions also contain cis-acting RNA elements important for HCV RNA replication and regulation (40,63). Last, the newly synthesized HCV proteins and genomic RNA are packaged to form progeny virus particles. However, the molecular aspects underlying HCV virion assembly, matur...

Hepatitis c virus and host cell lipids: An intimate connection

“…However, both kinds of particles are tightly associated with other lipoproteins, most notably ApoE and ApoC1. 174,175 These discrepancies are probably due to a defect of Huh7 cells to secrete ApoBcontaining VLDL particles, 173 supporting the notion of 'lipid imprinting' of HCV particles by the host cell.…”

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].…”

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

“…First, alteration of the lipoprotein pathway by inhibition of the microsomal triglyceride transfer protein (MTP) or of the diacylglycerol acyltransferase-1 (DGAT-1) or silencing of apoB or apoE expression decreases the production of infectious HCVcc virions. [12][13][14] Second, the phospholipid compositions of HCVcc and TRL share similar characteristics, whereas they strikingly differ from those of cellular membranes or envelopes of virus that assemble at cellular membranes. [15][16][17] Furthermore, lipoprotein lipases that specifically hydrolyse lipoprotein triacylglycerol modify HCVcc biochemical and physical features and decrease their infectivity.…”