丙型肝炎病毒（HCV）是一种正链RNA病毒，主要引起慢性肝炎、肝硬化和肝细胞癌。最近我们证实了 HCV RNA 基因组 NS5A 基因内的 m5C 修饰。然而，m5C 修饰的作用及其与宿主蛋白的相互作用在调节 HCV 生命周期中的作用仍有待探索。在这里，我们证明 HCV 感染通过转录因子 MAX 增强宿主 m5C 阅读器 YBX1 的表达。 YBX1充当m5C阅读器，识别HCV RNA基因组中m5C修饰的NS5A C7525位点，并显着增强HCV RNA稳定性。 YBX1 与脂滴和 HCV 核心蛋白的共定位也需要这种 m5C 修饰。此外，YBX1 促进 HCV RNA 复制以及病毒组装/出芽。 YBX1 65 位 (W65) 的色氨酸残基对于这些功能至关重要。 YBX1 的敲除或 YBX1 抑制剂 SU056 的应用可抑制 HCV RNA 复制和病毒蛋白翻译。据我们所知，这是第一份证明宿主 m5C 阅读器 YBX1 和 HCV RNA m5C 甲基化之间的相互作用促进病毒复制的报告。因此，肝脏 YBX1 敲低有望成为 HCV 治疗的潜在宿主导向策略。

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.