Persistent expression of hepatitis C virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells

Abstract

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.

Figure 1. Immunogold localization of HCV proteins and alteration of ultrastructure in cells expressing HCV proteins.

A, immunogold EM analyses. (i), Core proteins on the inner membrane of mitochondria; (ii), NS5A in the Golgi; (iii), NS5A in the matrix and inner membrane of mitochondria; (iv), NS5B in the mitochondria (binding to the outer membrane, inner membrane and the matrix) and rough endoplasmic reticulum (RER). Gold particles  = 15 nm. B, transmission electron microscopy analysis of ultrastructure. (i) Huh7 cells with compact, dense mitochondria (M) and RER; (ii) a representative image from HCV genome-length replicon cells with enlarged mitochondria (M) with focal loss of cristae, accumulation of lipid droplets (L), focally dilated RER (arrow head), and the presence of autophagocytic vacuoles (arrow) and double membrane vesicles (DMV).

Figure 2. Mitochondrial defects associated with the presence of HCV proteins.

A, mitochondrial sizes determined from electron micrographs from different HCV protein-expressing Huh7 cells; B, average number of mitochondria per cell. C, MitoSOX staining intensity quantified as average pixel intensity per cell; D, images of MitoSOX staining showing increased ROS production in the mitochondria of genome-length replicon, subgenomic replicon, and Core-on cells. E, MitoTracker Green stain showing the density and distribution of mitochondria. Nuclei are stained with Hoechst. Data in B and C are presented as mean ± SEM. A minimum of 40 cells each were analyzed.

Figure 3. Enhanced steady-state autophagy in genome-length and subgenomic replicon cells.

A, a representative EM image from subgenomic replicon cells showing the presence of autophagocytic vacuoles (A) and autophagocytic vesicles (a). Enlarged mitochondria (M) are also evident in this image. B, autophagosome counts per cell. A minimum of 12 cells each were analyzed. Data are presented as mean ± SEM. *, p<0.0001. C, immunocytochemical staining of LC3. The pixilated staining pattern of LC3, which is an indication of autophagosome formation and maturation, is consistently observed in genome-length and subgenomic replicon cells. Huh7 treated with Bafilomycin (Baf) was used as a positive control. D, western blot quantification of LC3-II. The upper panel shows a representative western blot result and the lower panel shows the quantification of LC3-II levels. Total LC3 levels (LC3-I + LC3-II) were also significantly increased in subgenomic replicon cells (p = 0.0028) and Huh7 cells treated with Bafilomycin (p = 0.0278). Data are presented as mean ± SEM of four independent experiments. LC3 levels were normalized to that of β-actin. #, p<0.05.

Figure 4. Redox state affects HCV-mediated autophagy.

Total LC3 (LC3-I + LC3-II) and LC3-II levels were quantified by western blot analyses. A, Dual expression vectors for increasing cytosolic (SOD1/cCAT) or mitochondrial (SOD2/mCAT) antioxidant expression were used, and mock-transfected cells served as controls. Cells were analyzed 36 hrs after the initial transfection. B, Xanthine (X) and xanthine oxidase (XO) were used to generate ROS to increased oxidative stress. To remove hydrogen peroxide and leaving only superoxide in the X/XO system, catalase was added to the reaction (X/XO/CAT). Cells were treated for 72 hrs. Data are presented as mean ± SEM of three independent experiments. LC3 levels were normalized to that of β-actin. Significant results from one-way ANOVA are shown. *, p<0.05 when compared to mock or no treatment controls.

Figure 5. Antioxidant profiles in HCV protein-expressing cells.

Protein levels of antioxidant enzymes in the cytosol (left panels) and mitochondria (right panels) were determined by western blot analyses. Data are presented as mean ± SEM of three (PRDX and TRX) or four (SOD) independent experiments. All protein levels were normalized to that of β-actin.

Figure 6. Population doubling time (A) and cyclin D1 level (B) in HCV protein-expressing Huh7 cells.

Data are presented as mean ± SEM of 4–5 (population doubling time) or three (cyclin D1) independent experiments.

Figure 7. Diagrammatic presentation of the cytopathic effects caused by various HCV proteins.

Replication of HCV RNA and production of HCV structural (core, E1, E2, and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) in the rough ER and the subsequent partition of HCV proteins to different subcellular compartments lead to ER stress, mitochondrial injury, and the production of ROS. These cytopathic effects lead to activation of autophagy without a concomitant increase in protein degradation. Consequently, autophagosomes accumulate in HCV infected cells. EM photo with normal mitochondria (M) and rough ER (RER) was taken from healthy Huh7 cells; EM photos with enlarged mitochondria, ER blebbing, lipid droplet (L), and autophagosomes (A) were taken from genome-length and subgenomic replicon cells. The HCV genetic materials contained in genome-length replicon, subgenomic replicon, and Core-on cells are depicted at the top of the diagram.