Zhu N, Khoshnan A, Schneider R, Matsumoto M, Dennert G, Ware C, Lai M M
Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles 90033, USA.
J Virol. 1998 May;72(5):3691-7. doi: 10.1128/JVI.72.5.3691-3697.1998.
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.
丙型肝炎病毒（HCV）核心蛋白除了作为病毒核衣壳的组成成分外，还是一种多功能蛋白。此前，我们已经表明核心蛋白可与淋巴毒素β受体的胞质结构域结合，淋巴毒素β受体是肿瘤坏死因子受体（TNFR）家族的成员。在本研究中，我们证明核心蛋白还可与TNFR 1的胞质结构域结合。体外通过谷胱甘肽S-转移酶融合蛋白下拉试验以及体内通过膜浮选法均证实了这种相互作用。体内和体外结合均需要TNFR 1的345至407位氨基酸残基，这对应于该受体的“死亡结构域”。我们进一步表明，在小鼠细胞系（BC10ME）或人细胞系（HepG2和HeLa细胞）中稳定表达核心蛋白会使它们对TNF诱导的凋亡敏感，这通过TNF细胞毒性或膜联蛋白V凋亡试验得以确定。核心蛋白的存在并未改变细胞中TNFR 1 mRNA的水平或细胞表面TNFR 1的表达，这表明病毒核心蛋白使细胞对TNF敏感并非由于TNFR 1的上调。此外，我们观察到核心蛋白可阻断TNF诱导的鼠BC10ME细胞中RelA/NF-κB的激活，从而至少部分解释了BC10ME细胞对TNF敏感性增加的原因。然而，在表达核心蛋白的HeLa或HepG2细胞中NF-κB的激活并未被阻断，这意味着核心蛋白使TNF敏感的另一种机制。这些结果共同表明，核心蛋白可能通过与TNFR 1的胞质尾部相互作用，经由TNF信号通路在HCV感染期间促进细胞死亡。因此，TNF可能在HCV发病机制中发挥作用。
