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. 2017 Nov 6;18(11):2346.
doi: 10.3390/ijms18112346.

Modulation of Cell Death Pathways by Hepatitis C Virus Proteins in Huh7.5 Hepatoma Cells

Olga V Masalova  1 , Ekaterina I Lesnova  2 , Pavel N Solyev  3 , Natalia F Zakirova  4 , Vladimir S Prassolov  5 , Sergey N Kochetkov  6 , Alexander V Ivanov  7 , Alla A Kushch  8
Affiliations

Modulation of Cell Death Pathways by Hepatitis C Virus Proteins in Huh7.5 Hepatoma Cells

Olga V Masalova et al. Int J Mol Sci. .

Abstract

The hepatitis C virus (HCV) causes chronic liver disease leading to fibrosis, cirrhosis, and hepatocellular carcinoma. HCV infection triggers various types of cell death which contribute to hepatitis C pathogenesis. However, much is still unknown about the impact of viral proteins on them. Here we present the results of simultaneous immunocytochemical analysis of markers of apoptosis, autophagy, and necrosis in Huh7.5 cells expressing individual HCV proteins or their combinations, or harboring the virus replicon. Stable replication of the full-length HCV genome or transient expression of its core, Е1/Е2, NS3 and NS5B led to the death of 20-47% cells, 72 h posttransfection, whereas the expression of the NS4A/B, NS5A or NS3-NS5B polyprotein did not affect cell viability. HCV proteins caused different impacts on the activation of caspases-3, -8 and -9 and on DNA fragmentation. The structural core and E1/E2 proteins promoted apoptosis, whereas non-structural NS4A/B, NS5A, NS5B suppressed apoptosis by blocking various members of the caspase cascade. The majority of HCV proteins also enhanced autophagy, while NS5A also induced necrosis. As a result, the death of Huh7.5 cells expressing the HCV core was induced via apoptosis, the cells expressing NS3 and NS5B via autophagy-associated death, and the cells expressing E1/E2 glycoproteins or harboring HCV the replicon via both apoptosis and autophagy.

Keywords: apoptosis; autophagy; caspase; hepatitis C virus; hepatoma Huh7.5 cells; necrosis; replicon.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Several hepatitis C virus (HCV) proteins hamper growth/viability of Huh7.5 cells. The cells, transfected with plasmids encoding HCV proteins, were counted 72 h posttransfection by microscopy visualization, and the values were normalized to the number of cells transfected with the empty pcDNA3.1(+) vector. The data are presented as means ± standard error of the mean (SEM) of eight measurements done in three independent experiments. * p < 0.05 vs. cells transfected with pcDNA3.1(+) vector (black bar).
Figure 2
Figure 2
HCV proteins affect activation of caspases-3, -8 and -9 in Huh7.5 cells in different manners. (a) Immunofluorescent staining of the activated caspase-9 and HCV proteins in Huh7.5 cells transiently expressing the HCV core or NS5A proteins, or harboring the full-length HCV replicon (400× magnification). Vertical panels left to right: staining with rabbit anti-caspase-9 primary and anti-rabbit secondary antibodies conjugated to Cy3 (orange), merge with nuclear staining with DAPI (blue), staining with mouse monoclonal antibodies to HCV proteins and anti-mouse secondary antibodies conjugated to fluoresceine isothiocianate (FITC; green), combined with nuclear staining with DAPI (blue). The arrows indicate caspase-9 positive cells. (b–d) Percentages of the cells which tested positive for the caspases-9 (b), -3 (c), and -8 (d). Values on each diagram are means ± SEM of eight measurements done in three independent experiments, * p < 0.05 compared to the cells transfected with the empty vector (black bar).
Figure 3
Figure 3
The HCV core and E1/E2 increase the number of Huh7.5 cells with nuclear DNA fragmentation, i.e., at the end stage of apoptosis. (a) Huh7.5 cells transfected with the core- and E1/E2-expressing plasmid or the empty pcDNA3.1 vector were stained 72 h posttransfection with the “DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System” kit (green), with mouse monoclonal antibodies on HCV proteins and anti-mouse secondary antibodies conjugated to Alexa Fluor 594 (AF594), and with DAPI. Vertical panels left to right: TUNEL staining (green), HCV proteins (red), and overlay of TUNEL, HCV proteins and DAPI staining; (b) Percentages of TUNEL-positive cells. Values are means ± SEM of eight measurements done in three independent experiments, * p < 0.05 compared to the cells transfected with the empty vector (black bar).
Figure 4
Figure 4
HCV proteins activated autophagy in Huh7.5 cells, as revealed by the enhanced incorporation of monodansylcadaverine into autophagosomes and detection of LC3. (a) Huh7.5 cells harboring the HCV replicon, or transfected with the E1/E2-expressing plasmid or the empty pcDNA3.1(+) vector were stained 72 h posttransfection with the monodansylcadaverine (MDC) and with DAPI. Vertical panels from the left to the right are: MDC staining (green), nuclear staining with DAPI (blue), overlay of MDC and DAPI staining; (b) Percentages of MDC-positive cells. (c) immunofluorescent staining of the activated LC3 and NS5B protein in Huh7.5 cells (400× magnification). Vertical panels left to right: staining with rabbit anti-LC3 primary and anti-rabbit secondary antibodies conjugated to Cy3 (orange) or primary mouse anti-NS5B antibody and secondary anti-mouse antibodies conjugated to FITC (green), merge with nuclear staining with DAPI (blue) The arrows indicate cells with LC3 punctuate staining; (d) Percentages of LC3-positive cells. Values are means ± SEM of eight measurements done in three independent experiments, * p < 0.05 compared to the cells transfected with the empty vector (black bar).
Figure 5
Figure 5
HCV NS5A protein promotes necrotic cell death. Huh7.5 cells harboring the HCV replicon, or transfected with the HCV-expressing plasmid or the empty pcDNA3.1(+) vector were stained 72 h posttransfection with trypan blue, and the stained cells were counted. The bars represent percentages of trypan-positive cells. Values are means ± SEM from three independent experiments, * p < 0.05 compared to the cells transfected with the empty vector (black bar).
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