Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial 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.

Bibliographic Details

Main Authors:	Queiroz,Sabrina R.A., Silva,Andréa N.M.R., Santos,Jefferson J.S., Marques Jr,Ernesto T.A., Bertani,Giovani R., Gil,Laura H.V.G.
Format:	Digital revista
Language:	English
Published:	Academia Brasileira de Ciências 2013
Online Access:	http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652013000100159
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