Oka Kiyomasa, Nagano-Fujii Motoko, Yoshida Isao, Hidajat Rachmat, Deng Lin, Akutsu Masato, Hotta Hak
Department of Microbiology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.
Microbiol Immunol. 2003;47(6):429-38. doi: 10.1111/j.1348-0421.2003.tb03380.x.
By using a vaccinia virus-T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c-Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2'-5'-oligoadenylate synthetase (2'-5'-OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein-expressing cells and the control, suggesting that the inhibition occurred at the post-transcription level. Pulse-labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C-terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C-terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full-length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.
通过使用痘苗病毒-T7表达系统，分析了丙型肝炎病毒（HCV）核心蛋白对培养细胞中瞬时表达的宿主细胞蛋白合成和积累的可能影响。免疫印迹和免疫荧光分析显示，某些核蛋白，如p21/Waf1、p53、增殖细胞核抗原和c-Fos的合成和积累受到HCV核心蛋白的强烈抑制。另一方面，细胞质蛋白，如2'-5'-寡腺苷酸合成酶（2'-5'-OAS）、RNase L和MEK1的合成和积累几乎不受HCV核心蛋白的影响。Northern印迹分析表明，表达HCV核心蛋白的细胞与对照之间这些蛋白的mRNA表达程度没有差异，这表明抑制发生在转录后水平。脉冲标记分析表明，HCV核心蛋白在翻译水平上强烈抑制p21/Waf1的合成。一旦在细胞核中积累，p21/Waf1的稳定性并未受到HCV核心蛋白的显著影响。C末端缺失18或41个氨基酸（aa）的HCV核心蛋白突变体几乎完全定位于细胞核中，失去了抑制p21/Waf1合成/积累的能力，而另一个C末端缺失8个aa的突变体仍保持与191个aa的全长HCV核心蛋白相同的特性（亚细胞定位和抑制作用）。综上所述，我们目前的结果表明，HCV核心蛋白在细胞质中的表达在翻译水平上选择性地抑制p21/Waf1和其他一些核蛋白的合成。
