Samarangenin B from Limonium sinense Suppresses Herpes Simplex Virus Type 1 Replication in Vero Cells by Regulation of Viral Macromolecular Synthesis

Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, samarangenin B (Sam B) was isolated from Limonium sinense; Sam B significantly suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of Sam B on HSV-1 replication was not due to the blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral immediate-early (α), early (β), and late (γ) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB), gC, gD, gG, and infected-cell protein 5 (ICP5) expression and gB mRNA expression in Vero cells were impeded by Sam B. Data from PCR showed that replication of HSV-1 DNA in Vero cells was arrested by Sam B. Furthermore, Sam B decreased DNA polymerase, ICP0, and ICP4 gene expression in Vero cells. Results of an electrophoretic mobility shift assay demonstrated that Sam B interrupted the formation of an α-trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of Sam B seem to be mediated, at least in part, by inhibiting HSV-1 α gene expression, including expression of the ICP0 and ICP4 genes, by blocking β transcripts such as DNA polymerase mRNA, and by arresting HSV-1 DNA synthesis and structural protein expression in Vero cells. These results show that Sam B is an antiviral agent against HSV-1 replication.

本研究考察了10种中草药乙醇提取物对1型单纯疱疹病毒（herpes simplex virus type 1, HSV-1）复制的抑制作用。通过生物活性导向分离（bioassay-guided fractionation）流程，从中华补血草（Limonium sinense）中分离得到Samarangenin B（Sam B）；实验结果显示，Sam B可在无明显细胞毒性的前提下，显著抑制维罗细胞（Vero）内HSV-1的增殖。
加药时序实验结果表明，Sam B对HSV-1复制的抑制作用并非通过阻断病毒吸附过程实现。为进一步定位Sam B阻断HSV-1复制周期的具体环节，本研究对介导病毒增殖的一系列关键调控事件进行了检测，涵盖病毒即刻早期（immediate-early, α）、早期（early, β）及晚期（late, γ）基因表达与DNA复制环节。
实验结果显示，维罗细胞内糖蛋白B（glycoprotein B, gB）、gC、gD、gG以及感染细胞蛋白5（ICP5）的表达水平，与gB mRNA的表达量均受到Sam B的显著抑制。PCR检测结果证实，Sam B可阻滞维罗细胞内HSV-1 DNA的复制过程。此外，Sam B可下调维罗细胞内DNA聚合酶、ICP0及ICP4的基因表达水平。
电泳迁移率变动分析（electrophoretic mobility shift assay）结果表明，Sam B可干扰α转录诱导因子/C1/Oct-1/GARAT多蛋白复合物的组装。
Sam B的抗病毒作用机制至少部分通过以下途径介导：抑制HSV-1 α基因（含ICP0与ICP4基因）的表达、阻断β类转录本（如DNA聚合酶mRNA）的生成、阻滞维罗细胞内HSV-1 DNA合成及结构蛋白的表达。上述研究结果证实，Sam B是一种可有效抑制HSV-1复制的抗病毒活性成分。