Maeda Naoyoshi, Furukawa Atsushi, Kakita Kosuke, Anada Masahiro, Hashimoto Shunichi, Matsunaga Shigeki, Kuroki Kimiko, Ose Toyoyuki, Kato Akihisa, Arii Jun, Kawaguchi Yasushi, Arase Hisashi, Maenaka Katsumi
Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences, Hokkaido University.
Biol Pharm Bull. 2016;39(11):1897-1902. doi: 10.1248/bpb.b16-00533.
Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILRα) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRα were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.
单纯疱疹病毒1型（HSV-1）是多种疾病的致病因子。尽管已经开发出如阿昔洛韦等抗疱疹药物，通过与DNA激酶相互作用来抑制病毒复制，但持续使用这些药物会导致耐药性HSV-1的频率增加，这是一个需要紧急解决的重要临床问题。最近，我们报道了源自HSV-1糖蛋白B（gB）蛋白的唾液酸化O-连接糖T抗原（sTn）及其连接的肽区域（O-糖基化sTn肽）在体外通过特异性靶向配对免疫球蛋白样2型受体α（PILRα）抑制HSV-1感染。在本研究中，为了进一步在体外鉴定gB介导的HSV-1感染的新型抑制剂，我们建立了一种基于细胞的融合试验用于快速药物筛选。将表达HSV-1 gB、gD、gH和gL以及T7 RNA聚合酶的表达质粒转染到中国仓鼠卵巢（CHO）细胞中，并将其指定为效应细胞。将稳定表达PILRα的CHO-K1细胞用T7启动子控制下的萤火虫荧光素酶表达质粒转染，并将其指定为靶细胞。将效应细胞和靶细胞共培养，当两种细胞成功融合时测量发光。重要的是，我们发现O-糖基化sTn肽以剂量依赖性方式特异性抑制细胞间融合。我们的结果表明，这种无病毒的基于细胞的融合试验系统可能是鉴定gB介导的HSV-1感染新型抑制剂的一种有用且有前景的方法，并将有助于开发针对HSV-1相关疾病的抗病毒治疗策略。
