Dataset Information

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

ABSTRACT: The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV₂ ), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV₇ , KIV₈ , and KIV₁₀ were not required.

Conclusions

Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851-1864).

SUBMITTER: Oliveira C

PROVIDER: S-EPMC5488163 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

Oliveira Catarina C Fournier Carole C Descamps Véronique V Morel Virginie V Scipione Corey A CA Romagnuolo Rocco R Koschinsky Marlys L ML Boullier Agnès A Marcelo Paulo P Domon Jean-Marc JM Brochot Etienne E Duverlie Gilles G Francois Catherine C Castelain Sandrine S Helle Francois F

Hepatology (Baltimore, Md.) 20170428 6

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethyle ...[more]

PMID: 28152568

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Project description: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.The 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.

Project description:A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

Dataset Information

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

Conclusions

Publications

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

Similar Datasets

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