Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus

Abstract

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.

FIG. 1.

Expression of HCV proteins in HCV-infected cells. (A) Analysis of the expression of HCV proteins C, E1, E2, and NS3 by Western blotting. Lysates of naive (−) or HCV-infected (+) cells were separated by SDS-PAGE and revealed by Western blotting with MAbs ACAP27 (anti-C), 1C4 (anti-E1), 3/11 (anti-E2), and 486D39 (anti-NS3). The sizes of protein molecular mass markers are indicated on the right (in kilodaltons). (B) Analyses of the glycans associated with HCV glycoprotein E2. Lysates of HCV-infected cells Huh-7 cells (JFH1) or 293T cells transfected with a plasmid expressing HCV envelope glycoproteins (phCMV-E1E2) were immunoprecipitated with anti-E2 MAb AP33. The immunoprecipitates were treated or not treated with endo H or PNGase F. Proteins were then separated by SDS-PAGE and revealed by Western blotting with the anti-E2 MAb 3/11.

FIG. 2.

Confocal immunofluorescence analysis of the intracellular distribution of HCV glycoproteins. Infected cells grown on coverslips were fixed and processed for double-label immunofluorescence for E1, E2, and the following cellular markers: calnexin, a chaperone of the ER; GM130, a Golgi matrix protein; ERGIC-53, a marker of the ER-to-Golgi intermediate compartment; or LAMP-1, a marker of late endosomes and lysosomes. Anti-E2 mouse MAb AP33 was used for the colocalization with E1. For the other experiments, E2 was revealed with the rat MAb 3/11. Representative confocal images of individual cells are shown with the merge images in the right column. Bar, 20 μm.

FIG. 3.

Cell surface expression of HCV glycoprotein E2. Naive (B and D) or infected (A and C) Huh-7 cells grown on coverslips were labeled with the anti-E2 MAb 3/11. A set of cells was fixed with 3% paraformaldehyde and processed for immunofluorescence labeling after permeabilization with Triton X-100 (A and B). Cell surface labeling (C, D, E, and F) was carried out on ice with MAb 3/11 before the fixation with 3% paraformaldehyde and the incubation with Alexa488-labeled secondary antibody. Huh-7 cells transfected with a plasmid expressing wild-type HCV envelope glycoproteins (phCMV-E1E2; panel E) or HCV envelope glycoproteins containing a mutation of the charged residues in the transmembrane domain of E2 [phCMV-E1E2(LAL); panel F] were used as controls of cell surface expression of E2. All images were acquired and processed with the same settings. Bar, 50 μm.

FIG. 4.

Confocal immunofluorescence analysis of the intracellular distribution of the capsid protein. Infected cells grown on coverslips were fixed and processed for double-label immunofluorescence for C (green) and the following cellular markers (red): ERGIC-53, a marker or the ER-to-Golgi intermediate compartment; GM130, a Golgi matrix protein; calnexin, a chaperone of the ER; or ADRP, a marker of the cytosolic pool of lipid droplets. Lipid droplets were stained with oil red O. Representative confocal images of individual cells are shown with the merge images in the right column. Insets display zoomed views of the indicated area. Bar, 20 μm.

FIG. 5.

Relative intracellular localization of HCV proteins C, E2, and NS3 analyzed by confocal immunofluorescence. Infected cells grown on coverslips were fixed and processed for double-label immunofluorescence for C and E2 (top row), NS3 and C (middle row), or NS3 and E2 (bottom row). Lipid droplets were stained with oil red O. Representative confocal images of individual cells are shown with the merge images in the right column. Insets display zoomed views of the indicated area. Bar, 20 μm.

FIG. 6.

Membrane alterations in Huh-7 cells containing replicative JFH1 genome visualized by electron microscopy. (A) Cell containing several lipid droplets (arrows). Bar, 2 μm. (B) Low-power overview showing a membranous web (arrow). Bar, 2 μm. (C) Higher magnification of the membranous web. Bar, 0.5 μm. (D) Small cisternae containing electron-dense elements compatible in size and shape with virus-like or core particles (arrows). Bar, 0.2 μm. Inset displays zoomed view of the indicated area.