Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

Abstract

The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra-and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCEThe HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles but likely contributes to the quality control of secreted infectious virions. These results shed new light on the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE.

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

“…apoE association. 26,31 If apoE protects HCV glycoproteins from nAbs directly, then our observations regarding the nAb-sensitivity of HCV generated from apoE knockdown cells will not be mediated by a marked difference in buoyant density distribution. To test this hypothesis, we analyzed the buoyant density distribution of infectious particles using isopycnic density gradient ultracentrifugation.…”

“…26,31 To define E2 domains relevant for association with apoE and neutralization escape, we took advantage of a mutation identified in variant A (VA), a pretransplant variant that was selected against in the same transplant recipient that contained the predominant VL variant. 6 Indeed, replacing phenylalanine with the leucine encoded in VA rendered the virus both sensitive to neutralization and less dependent on CD81 for cell entry.…”

“…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. 26 …”

Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

“…Cell culture-derived HCV (HCVcc) as well as LVPs can also be immunoprecipitated with antibodies against apoE and apoB (8 -10). Moreover, a direct interaction between apoE and viral envelope glycoproteins has recently been demonstrated (11)(12)(13), further indicating a close relationship between viral particles and lipoprotein components.…”

<b>Pol I-transcribed hepatitis C virus genome RNA replicates, produces an infectious virus and leads to severe hepatic steatosis in transgenic </b><b>mice </b>

“…As the apoE protein is an essential factor for the production of HCV particles, only the liver is expected to produce an infectious virus via the apoE protein (2,7,13,26).…”