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

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.

The Role of Bcl-xL Protein in Viral Infections

“…Scientific findings suggest that the pathogenic processes promote the death of infected cells. Apart from necrosis and autophagy, hepatocyte apoptosis plays a significant role in the development of liver disease [ 54 ]. Javed and Manzoor [ 53 ] have examined levels of two Bcl-2 family members, anti-apoptotic Bcl-xL and pro-apoptotic Bax, in Huh-7 cells (hepatocytes derived from the cellular carcinoma cell line) transfected with NS4A and NS3-NS4A of HCV genotype 3a.…”

Genetically Modified Mouse Mesenchymal Stem Cells Expressing Non-Structural Proteins of Hepatitis C Virus Induce Effective Immune Response

“…Expression of HCV proteins in the transfected MSCs was determined by the methods of indirect immunofluorescence and immunoperoxidase staining, using monoclonal antibodies (mAbs) against HCV proteins [33] as primary antibodies and secondary antibodies against mouse immunoglobulins (Ig) conjugated with fluorescein isothiocyanate (FITC) or horseradish peroxidase (HRP) (Dako, Denmark), as previously described [34,35]. Cell nuclei were stained with 4 -6-diamidino-2-phenylindole (DAPI) (immunofluorescence analysis) or with hematoxylin (immunoperoxidase method).…”

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus

“…The plasmid was purified from E. coli strain JM109 using a commercial QIAGEN Plasmid Purification Maxi Kit (QIAGEN, Hinden, Germany) according to the manufacturer’s instructions. To confirm the plasmid functionality, Huh7.5 cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific, Rockford, IL, USA), as described above [ 69 ]. MSCs of mice were transfected with the same plasmid using Xfect Transfection Reagent (Clontech Laboratories, Takara, San Jose, CA, USA), and a stably transfected MSC (mMSC) line was obtained using the G-418 selective antibiotic, as described earlier [ 17 ].…”