Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

<div><p>Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.</p></div

Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus

<div><p>Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV.</p></div

Amphipathic α-helices in apolipoproteins participate in the infectious particle formation of HCV.

<p>(A) Predicted or experimentally determined secondary structures of apolipoproteins. Secondary structures of the helices and sheets in the apolipoproteins are colored red and cyan, respectively. The three-dimensional structures of ApoA1 (Protein Data Bank (PDB) ID, 3R2P), ApoC1 (PDB ID, 1IOJ), ApoD (PDB ID, 2HZR), ApoE (PDB ID, 2L7B), ApoH (PDB ID, 1C1Z) and ApoM (PDB ID, 2XKL) are also shown in a ribbon model using the same color code of secondary structures. In cases in which the structure was not available, the secondary structure was predicted by using a CLC Genomics Workbench. (B,C) Schematics of the ApoE- and ApoC1-deletion mutants (upper). Deletion mutants with HA tags expressed in BE-KO1 cells by lentiviral vectors were detected by immunoblotting (middle). BE-KO1 cells expressing the WT or deletion mutants of ApoE or ApoC1 were infected with HCVcc at an MOI of 1, and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. Asterisks indicate significant differences (**, P<0.01) versus the results for control cells. (D) Schematic of the concentration of viral particles from HCV-infected cells using ultracentrifugation. (E) BE-KO1 cells expressing the WT or deletion mutants of ApoE were infected with HCVcc at an MOI of 1. Culture supernatants harvested at 72 h post-infection were concentrated by ultracentrifugation at 32,000 rpm for 2 h at 4°C, and subjected to immunoblotting.</p

Several apolipoproteins participate in HCV propagation.

<p>(A) Relative mRNA expression of the apolipoproteins in the liver tissues (left columns) was determined using the NextBio Body Atlas application. The median expression (right columns) was calculated across all 128 human tissues from 1,068 arrays using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. mRNA expression for each gene was log10 transformed. (B) Log10 transformed, normalized signal intensity of the apolipoproteins in Huh7 (left columns) and HepG2 (right columns) cells were extracted from previously published expression microarray dataset GSE32886. (C) Huh7 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA2 (A2), ApoB (B), ApoE (E) and control (Cont), and expression levels of apolipoproteins (upper panel) and infectious titers in the culture supernatants (lower panel) were determined by immunoblotting and a focus-forming assay at 72 h post-infection, respectively. (D) ApoA1, ApoA2, ApoC1, ApoE and ApoH were exogenously expressed in control and ApoE-knockdown Huh7 cells by lentiviral vectors. Expressions of the apolipoproteins were determined by immunoblotting analysis. (E) Infectious titers in the culture supernatants of control and ApoE-knockdown Huh7 cells expressing the apolipoproteins were determined by focus-forming assay at 72 h post-infection. In all cases, asterisks indicate significant differences (*, P<0.05; **, P<0.01) versus the results for control cells.</p

Accumulation of core proteins around lipid droplets in BE-KO1 cells.

<p>(A) Extracellular and intracellular infectious titers in Huh7, BE-KO1 and ApoE-restored cells infected by lentiviral vector (ApoE-res) were determined at 72 h post-infection with HCVcc at an MOI of 1 by focus-forming assay. Asterisks indicate significant differences (**, P<0.01) versus the results for parental cells. (B) BE-KO1 cells infected with HCVcc at an MOI of 1 were stained with anti-Core antibody at 72 h post-infection and examined by fluorescence microscopy. Identical fields were observed under electron microscopy by using the correlative FM-EM technique. The boxed areas are magnified and displayed. Huh7, BE-KO1 and ApoE-res cells infected with HCVcc at an MOI of 1 were subjected to immunofluorescence analyses by using anti-Core antibody (C), and immunoblotting by using antibodies against Core, NS3, ApoE, and actin at 72 h post-infection (D). Lipid droplets and cell nuclei were stained by BODIPY and DAPI, respectively. (E) BE-KO1 and ApoE-res cells infected with Jc1 strain-based HCVcc (HCVcc/Jc1; left panel) or JFH1 strain-based HCVcc (HCVcc/JFH1; right panel) at an MOI of 1 were subjected to immunofluorescence analysis by using anti-Core antibody at 72 h post-infection. Lipid droplets and cell nuclei were stained by BODIPY and DAPI, respectively.</p

Apolipoproteins participate in the post-envelopment step of the HCV life cycle.

<p>The supernatants (A) and lysates (B) of BE-KO1 and ApoE-restored (ApoE-res) cells infected with HCVcc at an MOI of 1 were subjected to density gradient fractionation. Each fraction was subjected to immunoblotting using anti-Core antibody (upper). The infectious titers and densities of each fraction were determined (lower). (C) The lysates of BE-KO1 and ApoE-res cells infected with HCVcc at an MOI of 1 were subjected to proteinase K digestion protection assay. Lysates were separated into 3 parts and incubated for 1 h on ice in the presence or absence of 50 µg/ml proteinase K with/without pretreatment with 5% Triton-X and then subjected to immunoblotting.</p

SR-B1 and LDLR have a redundant role in HCV entry.

<p>(A) Expressions of SR-B1 and LDLR in parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells were determined by immunoblotting analysis (upper panel). Cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (lower panel). (B) Parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels were determined at 24, 48 and 72 h post-infection by qRT-PCR. (C, D) Fluorescence localizations in parental, SR-KO, LD-KO and SR/LD-KO Huh7 cells were observed with a confocal microscope, upon infection with HCVcc at an MOI of 1 at 12, 24, 36, 48 and 60 h post-infection. The frequency is shown as the ratio of infected cells to total cells. (E) SR-B1 and LDLR were exogenously expressed in parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells by infection with lentiviral vectors. Expressions of SR-B1 and LDLR in these cells were determined by immunoblotting analysis (left panel). Cells were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (right panel). (F) SR-B1 and LDLR were exogenously expressed in SR/LD-DKO Huh7 cells by infection with different amount of lentiviral vectors. Expressions of SR-B1 and LDLR in these cells were determined by immunoblotting analysis (upper panel). Cells were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (lower panel). Asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for control cells.</p

MTTP participates in the formation of infectious HCV particles through the maturation of ApoB.

<p>(A) Expressions of ApoB, ApoE and MTTP in Huh7, B-KO1, M-KO1, E-KO1, BE-KO1 and EM-KO1 cells were determined by immunoblotting analysis. Cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA (B) and infectious titers in the supernatants (C) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively. The expressions of ApoB, ApoE and MTTP in BE-KO1 and EM-KO1 cells with/without expression of ApoE or MTTP by lentiviral vector were determined by immunoblotting (D) and ELISA (E). Cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA (F) and infectious titers in the supernatants (G) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively.</p

Density-dependent entry of LVPs via lipoprotein receptors.

<p>HCV particles in the culture supernatants of Huh7.5.1 cells were harvested at 72 h post-infection with HCVcc and fractionated by using density gradient centrifugation. Infectious titers of each fraction were determined by focus-forming assay and the buoyant density was plotted for each fraction (upper panels). SR/LD-DKO Huh7 cells expressing either SR-B1, LDLR or VLDLR were infected with each fraction and intracellular HCV RNA levels at 24 h post-infection were determined by qRT-PCR (lower panel). Asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for SR/LD-DKO Huh7 cells expressing SR-B1.</p

Host-derived apolipoproteins play comparable roles with viral secretory proteins E^rns and NS1 in the infectious particle formation of <i>Flaviviridae</i>

<div><p>Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the <i>Flaviviridae</i> members, pestivirus and flavivirus possess a viral structural protein E^rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while <i>Hepacivirus</i> including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of E^rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E^rns and NS1 in the formation of infectious particles. We examined whether E^rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either E^rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E^rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of E^rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of <i>Flaviviridae</i>, especially in the maturation of infectious particles, while E^rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.</p></div