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

The C-terminal amphipathic α-helix in E^rns is important to compensation for the role of apolipoproteins in the infectious particle formation of HCV.

<p>(A) The cDNA constructs for expression of HA-tagged E^rns (HA-E^rns) mutants; the full length of E^rns with the signal sequence of the C-terminal 30 amino acids of core protein (E^rns), and without the signal sequence (Δsig), the ectodomain of E^rns with the signal sequence (Ecto), and the C-terminal amphipathic α-helix of E^rns (Hel). (B) Expression of ApoE and the HA-E^rns mutants was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. (E) The insertion of two proline residues (204P/210P) in the Hel mutant containing the C-terminal amphipathic α-helix of HA-E^rns of CSFV was generated to examine the significance of the membrane-binding ability in the formation of HCV particles. Expression of Hel, Hel (204P/210P), and ApoE was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

Exchangeable apolipoproteins can compensate for the role of E^rns in the infectious particle formation of pestivirus.

<p>(A) Schematics of the wild type, E^rns deletion (CSFVΔE^rns), and polymerase dead (GAA) CSFV RNA, and the experimental procedure. (B) Expression of the full length of HA-tagged E^rns (HA-E^rns), HA-ApoA1, HA-ApoC1, and HA-ApoE was determined by immunoblotting at 48-h post-transduction of lentiviruses into SK6 cells. Intracellular CSFV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-electroporation with CSFVΔE^rns by qRT-PCR and focus-forming assay, respectively. (E) Expression of HA-E^rns, Hel, and HA-ApoE was determined by immunoblotting at 48-h post-transduction of lentiviruses into SK6 cells. Intracellular CSFV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-electroporation with CSFVΔE^rns by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

ApoE, E^rns, and NS1 participate in the infectious particle formation.

<p>(A) Experimental procedure. (B) Expression of HA-tagged ApoE (HA-ApoE), the full length of HA-E^rns, the C-terminal amphipathic α-helix of HA-E^rns (Hel), and HA-NS1 from serotype 3 and 4 of DENV was determined by immunoblotting at 48-h post-transduction of lentiviruses into BE-KO cells. (C) Intracellular and extracellular infectious titers were determined at an MOI of 1 by a focus-forming assay. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO cells expressing HA-ApoE, HA-E^rns, Hel, and HA-NS1 at 72-h post-infection. (E) Experimental procedure. (F) Expression of the HA-tagged E^rns, Hel, and ApoE was determined by immunoblotting at 48-h post-transduction of lentiviruses into SK6 cells. (G) Extracellular CSFV RNA and infectious titers were determined at 72-h post-electroporation with CSFVΔE^rns RNA by qRT-PCR and focus-forming assay, respectively. Specific infectivity was calculated as extracellular infectious titers/extracellular CSFV RNA copies in SK6 cells. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

Flavivirus NS1 can compensate for the role of apolipoproteins in the infectious particle formation of HCV.

<p>(A) Gene structures of flavivirus and hepacivirus and the experimental procedure. (B) Expression of ApoE and HA-tagged NS1 (HA-NS1) from serotypes 1 to 4 of DENV was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. (E) Expression of ApoE and HA-NS1 from DENV, JEV, TBEV, and Yokose virus was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. (H) Expression of ApoE and HA-NS1 from DENV and JEV in 293T/CLDN1/miR-122 cells was determined by immunoblotting analysis. Cells were infected with HCV at an MOI of 10, and intracellular HCV RNA (I) and infectious titers in the supernatants (J) at 72-h post-infection were determined by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

NS1 can compensate for the role of E^rns in the infectious particle formation of CSFV.

<p>(A) Schematics of the wild type and E^rns deletion (CSFVΔE^rns) CSFV RNA, and the experimental procedure. (B) Expression of HA-tagged E^rns (HA-E^rns) and HA-NS1 from DENV and JEV was determined by immunoblotting at 48-h post-transduction of lentiviruses into SK6 cells. Intracellular CSFV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-electroporation with CSFVΔE^rns by qRT-PCR and focus-forming assay, respectively. (E) Expression of HA-E^rns, and the wild type and mutant (F160A and V162D) HA-NS1 from DENV was determined by immunoblotting. Intracellular CSFV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-electroporation with CSFVΔE^rns. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

E^rns can compensate for the role of apolipoproteins in the infectious particle formation of HCV.

<p>(A) Gene structures of flaviviruses, pestiviruses, and hepaciviruses, and the experimental procedure. (B) Expression of ApoE and HA-tagged E^rns (HA- E^rns) from BVDV, CSFV, and BDV was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-infection with JFH1 HCV at a multiple of infection (MOI) of 1 by qRT-PCR and focus-forming assay, respectively. (E) Expression of ApoE and HA-E^rns in 293T/CLDN1/miR-122 cells was determined by immunoblotting analysis. Intracellular HCV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-infection with JFH1 HCV at an MOI of 10 by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

NS1 mutants in the hydrophobic protrusion deficient in viral replication can compensate for the role of ApoE in the infectious particle formation of HCV.

<p>(A) Gene structure of a recombinant DENV with a luciferase gene and possessing a single amino-acid substitution in NS1 (F160A or V162D). (B) Luciferase activity was determined at 4, 24, and 48-h post-electroporation with the recombinant DENV RNA in BE-KO cells. (C) Experimental procedure. (D) Expression of ApoE and the wild type and mutant HA-tagged NS1 was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (E) and extracellular infectious titers (F) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* <i>p</i> < 0.01) versus the results of the control cells.</p

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

Lipid binding and lipid uptake of lipoprotein receptors participate in HCV entry.

<p>Schematics were shown for the SR-B1, LDLR and VLDLR mutants (upper panels of A and B, and left panel of C). (A) S112F- and T175A-SR-B1 were generated to examine the significance of lipid binding ability. (B) Seven or eight repeats in the ligand binding domains of LDLR (left) and VLDLR (right) were deleted (ΔLBD). (C) An asparagine residue in the repeat 5 and in the repeats 2 to 7 of LDLR was substituted with tyrosine (mut5 and mut2-7). The wild-type and mutants of SR-B1, LDLR and VLDLR were expressed in SR/LD-DKO Huh7 cells by lentiviral vectors. Expressions of these receptors were detected by immunoblotting (middle panels in A and B, right upper panel in C). These cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels at 24 h post-infection were determined by qRT-PCR (lower panels in A and B, right lower panel in C). (D) HCVpp were inoculated into parental, SR/LD-DKO expressing either SR-B1, LDLR or VLDLR and CD81-KO Huh7 cells, and luciferase activities were determined at 48 h post-infection by using a luciferase assay system. In all cases, asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for cells expressing wild-type receptors.</p