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

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

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

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

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

Characterization of miR-122-independent propagation of HCV

<div><p>miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV_122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV_122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.</p></div

Propagation of Con1C3/JFH_122KO in 751-122KO cells.

<p>(A) Infectious titer in the culture medium on serial passage of 751-122KO#1 or Huh7.5.1 cells. Red circles indicate the passage in 751-122KO cells, and the other circles indicate the passage in Huh7.5.1 cells. Three independent passages (#4–6, #4–8, #7–8) are shown. (B) Nuclear translocation of IPS-GFP (arrows) in Huh7.5.1 and 751-122KO cells upon infection with Con1C3/JFH and Con1C3/JFH_122KO. (C) Con1C3/JFH and Con1C3/JFH_122KO were inoculated into 751-122KO#1 and Huh7.5.1 cells, and the levels of intracellular HCV-RNA replication were determined. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control. (D) 293T-CLDN cells infected with either Con1C3/JFH or Con1C3/JFH_122KO were treated with IFNα and BILN and then the intracellular HCV-RNA level was determined at 12, 24 and 48 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control.</p