Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection.
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.
Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences, Hokkaido University.
Naoyoshi
N
Maeda
Atsushi
A
Furukawa
Kosuke
K
Kakita
Masahiro
M
Anada
Shunichi
S
Hashimoto
Shigeki
S
Matsunaga
Kimiko
K
Kuroki
Toyoyuki
T
Ose
Akihisa
A
Kato
Jun
J
Arii
Yasushi
Y
Kawaguchi
Hisashi
H
Arase
Katsumi
K
Maenaka
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000737:chemistry*; D000998:Antiviral Agents / Q000494:pharmacology*; D001681:Biological Assay; D016466:CHO Cells; D002459:Cell Fusion; D018920:Coculture Techniques; D006224:Cricetinae; D003412:Cricetulus; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D006561:Herpes Simplex / Q000188:drug therapy; D018259:Herpesvirus 1, Human / Q000276:immunology / Q000472:pathogenicity; D049409:Luciferases, Firefly / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D010455:Peptides / Q000494:pharmacology*; D011971:Receptors, Immunologic / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D000957:Antigens, Viral, Tumor; D000998:Antiviral Agents; D008562:Membrane Glycoproteins; C412263:PILRA protein, human; D010455:Peptides; D011971:Receptors, Immunologic; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C040495:glycoprotein B, Simplexvirus; D049409:Luciferases, Firefly; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases