Clathrin mediates infectious hepatitis C virus particle egress

Abstract

Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reductions in HCVcc secretion were observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments, and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its colocalization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route.

Importance: HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Here we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which may lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the very-low-density lipoprotein (VLDL) components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since, to date, it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism recently emerged as a potential target for therapies against HCV infection, our data may help in the design of new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective, and safe antivirals.

FIG 1

HCVcc egress is mediated by clathrin and AP-1. (A) HCV RNA and infectivity quantification after CHC and AP-1 knockdown in infected Huh7 cells. Results are expressed as means and standard errors of the means (SEM) for at least three experiments performed in triplicate. (B) Western blot analysis of CHC and AP-1 knockdown efficiencies in Huh7 cells. p53 was used as a loading control. (C) Western blot analysis of HCV core protein levels and CHC knockdown efficiency after transfection of HCVcc-infected Huh7 cells with either control siRNA, four different CHC-specific siRNAs, or the pooled CHC siRNA mix used for panel B. p53 was used as a loading control. Numbers indicate the relative intensities of bands compared to those for control cells after being normalized to p53 levels. (D) HCV RNA and infectivity quantification after CHC knockdown. Results are expressed as means and SEM for an experiment performed in triplicate. (E) Cell proliferation was assessed as described in Materials and Methods. Results obtained 48 h and 72 h after transfection are shown. (F) HCV RNA levels in a clone containing a full-length HCV replicon were analyzed by real-time RT-PCR. (G) HCV core levels were analyzed by Western blotting. p53 was used as a loading control. (H) HCV core levels were analyzed by immunofluorescence. Green, HCV core; blue (DAPI [4′,6-diamidino-2-phenylindole]), nuclei. (I) HCV RNA and infectivity quantification after CHC and AP-1 knockdown in HCVcc-producing Huh7 Lunet N cells. Results are expressed as means and SEM for at least three experiments performed in triplicate.

FIG 2

HCVcc secretion is impaired after clathrin and dynamin pharmacological inhibition. (A) HCV RNA levels after clathrin or dynamin inhibition in Huh7 Lunet N cells by 50 μM Pitstop 2 or 80 μM dynasore treatment, respectively. Data are presented relative to those for vehicle-treated control cells. Results are expressed as means and SEM for four experiments performed in triplicate. (B) Analysis of the possible effects of Pitstop 2 and dynasore carryover during titration of infective supernatants after inhibitor treatment. (C) Polarized hepatocyte-like localization of ZO-1 in Huh7 cells cultured on Matrigel. Red, ZO-1; blue (DAPI), nuclei. Bar, 25 μm. (D and E) Effects of Pitstop 2 (D) or dynasore (E) treatment on extracellular and intracellular HCV RNA levels in 3D-cultured, HCVcc-infected Huh7 cells. Results are presented relative to those for vehicle (dimethyl sulfoxide [DMSO])-treated control cells and are expressed as means and SEM for three experiments. (F and G) Western blot analyses of apoB and apoE levels in supernatants and cellular lysates obtained from 3D-cultured, HCVcc-infected Huh7 cells after clathrin (F) or dynamin (G) inhibition. α1AT and p53 were used as loading controls.

FIG 3

HCV exocytosis is not mediated by AP-2. (A) Western blot analysis of AP-2 knockdown efficiency. p53 was used as a loading control. (B) Immunofluorescence analysis of transferrin receptor localization after AP-2 knockdown. Bar, 25 μm. (C) HCVcc-infected cells were transfected with control or AP-2-specific siRNAs, and HCV RNA levels in supernatants were quantified by real-time RT-PCR. Results are presented relative to those for control siRNA-transfected cells and are expressed as means and SEM for six experiments performed in triplicate.

FIG 4

apoB and apoE secretion is not impaired by clathrin or AP-1 interference. (A and B) apoB and apoE levels were determined by ELISA (A) and Western blotting (B) after CHC or AP-1 knockdown in infected Huh7 cells. α1AT and p53 were used as loading controls. (C) apoB and apoE levels in the supernatants of infected Huh7 cells after 50 μM Pitstop 2 or 80 μM dynasore treatment were determined by ELISA. Results are expressed as means and SEM for two experiments performed in duplicate. (D and E) Kinetic analyses of secreted apoB, apoE, and α1AT (D) and of secreted HCV RNA (E) by Western blotting and real-time RT-PCR, respectively, after Pitstop 2 treatment of infected Huh7 cells. Results in panel E are presented relative to the maximum value and expressed as means and SEM for three experiments.

FIG 5

Intracellular HCVcc-apoE association in BFA-treated cells. (A) Immunoprecipitation (IP) from supernatants of HCVcc-infected cells, using control, anti-apoB, and anti-apoE antibodies. apoB and apoE levels were determined by Western blotting (WB). (B) Supernatant infectivity after immunoprecipitation was analyzed by titration on Huh7 cells and real-time RT-PCR. Results are presented relative to the infectivity of control Ig-immunoprecipitated supernatants and are expressed as means and SEM for three experiments. (C) Supernatant immunoprecipitation was carried out as described for panels A and B, and RNAs were extracted from both immunoprecipitates and postimmunoprecipitation supernatants. HCV RNA levels were assessed by real-time RT-PCR and presented as percentages of input HCV RNA. Results are expressed as means and SEM for three experiments. (D) Immunoprecipitates from detergent-free lysates of HCVcc-infected, siRNA-transfected cells were used for either Western blot analysis (inset; control siRNA-transfected cells) or HCV RNA quantification. (E) HCV RNA levels and infectivities after 1 μg/ml BFA treatment. (F) (Left) Immunoprecipitated HCV RNA levels after BFA treatment. (Right) Intracellular infectivities of BFA-treated cells after immunoprecipitation. (G) Intracellular infectivities of BFA-treated cells, assessed by titration on Huh7 cells in the presence of 5 μg/ml control or anti-apoE antibody. Results are expressed as means and SEM for at least three experiments.

FIG 6

Colocalization of HCV core with AP-1 and apoE after BFA treatment. HCVcc-producing Huh7 Lunet N cells were treated with 1 μg/ml BFA for 6 h and fixed for further immunofluorescence analysis. Green, HCV core; red, AP-1 (A) or apoE (B); blue (DAPI), nuclei. Images show a single x-y section from the confocal stack of a representative cell. The red box shows a detail of the merged image. Bars, 10 μm. In the scatterplots, each dot represents Mander's coefficient for HCV core (fraction of HCV core that colocalizes with AP-1 or apoE) for each of the 20 cells from two independent experiments (see Materials and Methods for details). The horizontal lines represent median Mander's coefficients for all cells studied.

FIG 7

Colocalization of HCV core with EEA1 and LAMP1 after CHC or AP-1 knockdown. (A) HCVcc-producing Huh7 Lunet N cells were transfected with control, CHC, or AP-1 siRNAs and fixed 2 days later for immunofluorescence analysis. Green, HCV core; red, EEA1 (left) or LAMP1 (right); blue (DAPI), nuclei. Images show a single x-y section from the confocal stack of a representative experiment. The red box shows a detail of the merged image. Bars, 10 μm. Quantification and scatterplot representations were performed as described in the legend to Fig. 6 and in Materials and Methods. (B) (Left) Western blot showing efficiency of CHC or AP-1 knockdown. (Right) Western blot showing unaltered expression levels of EEA1, LAMP1, and core after CHC or AP-1 knockdown. Numbers indicate the relative intensities of bands compared to those of control cells after being normalized to p53 levels.