Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection

Abstract

Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.

Fig. 1

Main ultrastructural changes encountered in Huh7.5 cells during the course of HCVcc infection (adapted JFH-1 strain). Cells displayed three types of membrane alterations: a, b vesicles in cluster (ViC), corresponding to a small number (10–40) of single-membrane vesicles of variable size (100–200 nm) grouped together in a well-delimited area; most, if not all, of these vesicles had an internal invagination (white arrow in the low magnification image in a; black arrow in an individual vesicle shown at high magnification in b). c, d Contiguous vesicles (CV), corresponding to single-membrane vesicles of a more homogeneous size (around 100 nm) present in large numbers; these vesicles clustered together and frequently formed a collar around lipid droplets (LD, see the white arrows on the high magnification image in d). e, f Double-membrane vesicles (DMV) of extremely variable size (150–1,000 nm); these vesicles were characterized by a thick, electron-dense membrane consisting of two closely apposed membranes (black arrows on the high magnification image in f). All these structures were highly specific to HCV infection and were not observed in uninfected Huh7.5 cells (not shown)

Fig. 2

Quantitative analysis of the various types of vesicle observed in Huh7.5 cells in the 6 days following HCVcc infection, and quantification of intracellular/extracellular HCV RNA. a Early in the time course, cells were infected on day 0 by incubation for 16 h with an optimized JFH-1 virus strain, then washed with medium and cultured for a further 3 days. b One flask was then treated with trypsin on day 2 to initiate a subculture which was continued until day 6. Each day, a cell pellet was collected for the qualitative and quantitative analysis, by EM, of 60 consecutive cell sections. ViCs were the least abundant structures and their frequency is indicated as the percentage (gray circles) of cell sections with at least one vesicle cluster. CVs and DMVs were extremely abundant in infected cells and their frequency is indicated as the percentage of cell sections containing no (0) vesicles, 1–50 vesicles, or more than 50 vesicles (light gray, dark grey, and black bars, respectively). Intracellular (white circles) and extracellular (black circles) HCV RNA were quantified at each time point, with the Abbott m2000sp–m2000rt real-time PCR assay. In all experiments, uninfected Huh7.5 cells were used as a negative control. The data presented are the mean values and standard deviations for three independent experiments

Fig. 3

Immuno-EM analyses of Huh7.5 cells on day 6 post infection with HCVcc, prepared by freeze substitution. a, b Immunogold labeling with a monoclonal antibody against core protein was observed over the entire surface of lipid droplets (LD) and on the membrane of the adjacent contiguous vesicles (CV). c, d Immunogold labeling with a monoclonal antibody against NS5A was more discrete than that for core protein, but was also observed in some areas of the LD surface (arrows in c), and on the membrane of the adjacent CVs (arrows in d). e Immunogold labeling with the monoclonal antibody against NS5A was also observed on double-membrane vesicle (DMV) membranes (arrows). f Immunogold labeling with the monoclonal antibody against dsRNA was observed within the DMVs or associated with DMV membranes (arrows). These observations were specific to Huh7.5-infected cells, as no immunogold labeling was detected in the uninfected Huh7.5 cells (not shown)

Fig. 4

Three-dimensional reconstruction of a whole Huh7.5 cell 2 days after infection with HCVcc. The standard EM block was resized for the cutting of a ribbon of 140 serial ultrathin sections (70 nm thick) to reconstruct this particular cell. Contours were drawn with IMOD software through the same specific cellular structures on different serial sections, including the plasma membrane (light gray), the nucleus (green), the lipid droplets (yellow), and the three types of membrane alterations specifically induced by HCV: ViCs (blue), CVs (purple) and DMVs (red). A QuickTime movie of the 3D reconstruction of this cell is also provided as supplemental material, to improve visualization of the spatial distribution of these structures within the cell