Karthe Juliane, Tessmann Kathi, Li Jisu, Machida Raiki, Daleman Maaike, Häussinger Dieter, Heintges Tobias
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany.
Hepatology. 2008 Sep;48(3):702-12. doi: 10.1002/hep.22366.
The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84-87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein-mediated pathogenesis.
丙型肝炎病毒（HCV）核心蛋白对于病毒基因组的包装至关重要，并且在脂肪变性、免疫逃逸和肝细胞癌中发挥重要作用。因此，它可能是干扰HCV生命周期及相关发病机制的一个有前景的治疗靶点。在本研究中，我们利用噬菌体展示技术从慢性丙型肝炎患者的骨髓浆细胞中产生针对核心蛋白的单链可变区抗体片段（scFv）。由此鉴定出一种具有高亲和力结合能力的抗体（scFv42C），其结合位点被定位到不同基因型间保守的核心蛋白的PLXG基序（第84 - 87位氨基酸残基）。当单独在Huh7人肝癌细胞系中表达时，scFv42C呈现弥漫性细胞质荧光，而与核心基因共转染则使其亚细胞分布转变为核心蛋白的亚细胞分布。荧光共振能量转移技术独立证实了scFv42C在细胞内与其靶标核心蛋白的结合。有趣的是，单链抗体的表达在细胞内降低了核心蛋白水平，特别是在完整HCV复制的情况下。此外，核心蛋白诱导的细胞增殖可通过scFv4C共表达来逆转。因此，scFv42C可能代表一种新型抗HCV药物，其作用机制是隔离核心蛋白并减轻核心蛋白介导的发病机制。
