A high-throughput assay using dengue-1 virus-like particles for drug discovery.
Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50-100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3x10(6)foci-forming unit/ml) is to use low temperature (30 degrees C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
Novartis Institute for Tropical Diseases, 10, Biopolis Road, #05-01 Chromos, Singapore, Singapore.
Min
M
Qing
Wei
W
Liu
Zhiming
Z
Yuan
Feng
F
Gu
Pei Yong
PY
Shi
D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003716:Dengue Virus / Q000187:drug effects* / Q000235:genetics; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D018274:Electroporation; D017930:Genes, Reporter; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008253:Macaca mulatta; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D022701:Virosomes / Q000187:drug effects* / Q000235:genetics
D000998:Antiviral Agents; D014764:Viral Proteins; D022701:Virosomes; D049410:Luciferases, Renilla