Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins

Abstract

Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells.

Methods: Seven truncated GFP (green fluorescent protein)-CORE fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191: 1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry.

Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa>1-126 aa>1-58 aa>127-172 aa>59-126 aa).

Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.

Fig. 1

Schematic representation of the open reading frame encoding various truncated CORE. Functional domains are shown in shadowed boxes along the diagrams. All of them fused to GFP N-terminal

Fig. 2

Schematic representation of different plasmid construction: Different truncated CORE were inserted into MCS: Nhe I and EcoR I to yield CORE-GFP fusion protein expression plasmids

Fig. 3

CORE-GFP localization in HepG2 cells. The confocal microscope setting for GFP detection was the same for all captured images: 488 nm excitation emission, laser power 10%, Iris1.8, Gain 75, background −10, emission filter 514/30. (a) CORE is only expressed in the cytoplasm associating with ER; (b) CORE is expressed not only in the cytoplasmic associating with ER but also in nuclear

Fig. 3

CORE-GFP localization in HepG2 cells. The confocal microscope setting for GFP detection was the same for all captured images: 488 nm excitation emission, laser power 10%, Iris1.8, Gain 75, background −10, emission filter 514/30. (a) CORE is only expressed in the cytoplasm associating with ER; (b) CORE is expressed not only in the cytoplasmic associating with ER but also in nuclear