Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea.
Ilsong Institute of Life Science, Hallym University, Anyang, 14066, Republic of Korea.
J Microbiol. 2021 Jan;59(1):101-109. doi: 10.1007/s12275-021-0470-8. Epub 2020 Dec 23.
Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.
丙型肝炎病毒 (HCV) 的生命周期高度依赖于细胞蛋白来进行病毒繁殖。为了鉴定参与 HCV 繁殖的细胞因子，我们之前使用 HCV 非结构 5A (NS5A) 蛋白作为探针进行了蛋白质微阵列分析。在微阵列中固定的约 9000 个人类细胞蛋白中，腺苷同型半胱氨酸酶样 1 (AHCYL1) 是鉴定为 NS5A 相互作用蛋白的 90 种蛋白之一。在这些候选物中，选择 AHCYL1 进行进一步研究。在本研究中，我们通过体外下拉和共免疫沉淀实验验证了 NS5A 与 AHCYL1 之间的物理相互作用。此外，HCV NS5A 与 Jc1 感染细胞中的内源性 AHCYL1 相互作用。在 HCV 复制细胞中，NS5A 和 AHCYL1 均共定位于细胞质区域。siRNA 介导的 AHCYL1 敲低可阻断 HCV 繁殖。外源性表达 siRNA 抗性 AHCYL1 突变体，但不表达野生型 AHCYL1，可恢复 HCV 蛋白表达水平，表明 AHCYL1 是 HCV 繁殖所必需的。重要的是，AHCYL1 参与 HCV 内部核糖体进入位点介导的 HCV 生命周期翻译步骤。最后，我们证明 AHCYL1 的蛋白酶体降解途径被持续性 HCV 感染所调节。总的来说，这些数据表明 HCV 可能调节 AHCYL1 蛋白以促进病毒繁殖。
