Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis

Abstract

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin beta receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the "death domain" of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-kappaB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-kappaB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.

FIG. 1

In vitro interaction of the HCV core protein with the cytoplasmic domain of TNFR 1 in a GST-binding assay. (a) Schematic representation of deletion and point mutants of GST-TNFR 1 (cytoplasmic domain) fusion proteins. The darkened region indicates the death domain. The numbers represent the starting and the ending amino acid residues of the TNFR 1 open reading frame in each construct. Binding of each protein to the HCV core protein is summarized at the right. (b) ³⁵S-labeled in vitro-translated HCV core protein (3 μl) was incubated with different GST-TNFR 1 fusion proteins. The bound core protein was separated by SDS-PAGE and detected by autoradiography. The input HCV core protein (1 μl; core probe) used was run in parallel. Shown is a representative result of at least three separate experiments.

FIG. 2

In vivo interaction between the HCV core protein and the cytoplasmic tail of TNFR 1 shown by membrane flotation analysis. Cos7 cells transfected with various plasmids were separated into membrane and cytosol fractions as described in Materials and Methods. The core protein and TNFR 1 were detected by immunoblotting with the appropriate antibodies. (a) The core protein expressed alone; (b) coexpression of the core protein with the full-length TNFR 1 (aa 1 to 426); (c) coexpression of the core protein and a C-terminus-truncated TNFR 1 (aa 1 to 308).

FIG. 3

Effects of HCV core protein expression on the sensitivity of various cell lines to TNF-induced cell death. Various stably transformed cell lines were treated with different concentrations of TNF or anti-TNFR 1 antibody as indicated for each experiment. Cells were assayed by the MTT assay (a to c) and by the annexin V assay (d). (a) BC10/CORE and control BC10/EF cells (n = 8 independent clones examined) incubated with murine TNF (mTNF). The inset shows the immunoblotting of the core protein in two representative BC10/EF and BC10/CORE clones. (b) HepG2/EF and HepG2/CORE cells (n = 4) incubated with anti-human TNFR 1 antibody. The inset shows the immunoblotting of the core protein in two representative HepG2/EF and HepG2/CORE clones. (c) HeLa/EF and HeLa/CORE cells incubated with human TNF (hTNF). The inset shows the immunoblotting of the core protein in HeLa/EF and HeLa/CORE cell pools. (d) HeLa/EF and HeLa/CORE cell clones incubated with human TNF in the annexin V apoptosis assay.

FIG. 4

Comparison of TNFR 1 mRNA levels in HeLa/EF and HeLa/CORE cell lines by Northern blotting. Approximately 20 μg of total RNA from each cell line was loaded in each lane. The same filter was used for hybridization with a TNFR 1 riboprobe and with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe.

FIG. 5

Comparison of cell surface TNFR 1 expression levels in core-expressing and control HepG2 cell lines by flow cytometry analysis. The cell surface TNFR 1 was detected by a goat anti-human sTNFR 1 (α-TNFRI) as the primary antibody and an FITC-conjugated mouse anti-goat IgG (α-IgG-FITC) as the secondary antibody. Cells exposed to the secondary antibody only (left panels) were used as negative controls. Cells are plotted against their FITC intensity, and the gated percentages of TNFR 1-positive cells of each cell line are shown in parentheses.

FIG. 6

Effect of HCV core protein expression on RelA activation by TNF. BC10/EF and BC10/CORE cells were treated with murine TNF (10 ng/ml, final concentration) for 20 min at 37°C and lysed with 0.5% Nonidet P-40. The nuclear (NE) and cytoplasmic (CE) fractions were used for immunoblotting analysis using antibodies specific for RelA, actin, or HCV core protein.