title: Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication.
abstract: Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a full-length DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5'noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.
authors.affiliation: Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. scvlk@mahidol.ac.th
authors.forename: Vijittra
authors.initials: V
authors.lastname: Leardkamolkarn
authors.forename: Wipawan
authors.initials: W
authors.lastname: Sirigulpanit
authors.forename: Nunya
authors.initials: N
authors.lastname: Chotiwan
authors.forename: Supeecha
authors.initials: S
authors.lastname: Kumkate
authors.forename: Claire Y-H
authors.initials: CY
authors.lastname: Huang
mesh_terms: D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012367:RNA, Viral / Q000378:metabolism*; D011995:Recombination, Genetic; D012093:Replicon; D013194:Staining and Labeling / Q000379:methods*; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D049452:Green Fluorescent Proteins