title: Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique.
abstract: RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potential for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.
authors: Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Universidade Federal de Pernambuco, 50760-420 Recife, PE, Brasil.
authors: Sabrina R A
authors: SR
authors: Queiroz
authors: Andréa N M R
authors: AN
authors: Silva
authors: Jefferson J S
authors: JJ
authors: Santos
authors: Ernesto T A
authors: ET
authors: Marques
authors: Giovani R
authors: GR
authors: Bertani
authors: Laura H V G
authors: LH
authors: Gil
mesh_terms: D003001:Cloning, Molecular; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D014779:Virus Replication; D015005:Yellow fever virus / Q000235:genetics* / Q000502:physiology
chemical_list: D012367:RNA, Viral