DataSheet_1_Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.docx

BackgroundAn increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target.
MethodsIn this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells.
ResultsThe dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2.
ConclusionWe established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

背景：功能性治愈需求的增长加速了慢性乙型肝炎新型治疗方法的研究，此类研究主要聚焦于恢复抗病毒免疫以控制病毒感染。此前，我们曾将延伸因子Tu GTP结合结构域包含蛋白2（EFTUD2）描述为一种先天免疫调节因子，并提出其可能作为抗病毒靶点。
方法：本研究构建了用于靶向EFTUD2化合物筛选的Epro-LUC-HepG2细胞模型。从261种免疫与炎症相关化合物中筛选出普乐沙福（Plerixafor）与瑞沙托维（resatorvid），因其可显著上调EFTUD2的表达。我们在HepAD38细胞以及感染乙型肝炎病毒（HBV）的HepG2-NTCP细胞中，检测了这两种化合物对HBV的作用效果。
结果：双荧光素酶报告基因实验显示，EFTUD2启动子hEFTUD2pro-0.5 kb的活性最强。在Epro-LUC-HepG2细胞中，普乐沙福与瑞沙托维可显著上调EFTUD2启动子的活性以及该基因的mRNA与蛋白表达水平。在HepAD38细胞以及感染HBV的HepG2-NTCP细胞中，两种化合物处理可呈剂量依赖性地强力抑制乙型肝炎表面抗原（HBsAg）、HBV DNA、HBV RNA以及共价闭合环状DNA（cccDNA）的水平。此外，恩替卡韦（entecavir）与任一化合物联合给药时，抗HBV效果可得到增强，而该作用可通过敲低EFTUD2得以阻断。
结论：本研究构建了一种便捷的靶向EFTUD2化合物筛选模型，并进一步在体外证实普乐沙福与瑞沙托维为新型HBV抑制剂。本研究结果为开发一类以宿主因子而非病毒酶为作用靶点的抗HBV药物提供了相关依据。