title: Construction and Rescue of a Functional Synthetic Baculovirus.
abstract: Synthetic viruses provide a powerful platform to delve deeper into the nature and function of viruses as well as to engineer viruses with novel properties. So far, most synthetic viruses have been RNA viruses (<30 kb) and small DNA viruses, such as bacteriophage phiX174. Baculoviruses contain a large circular dsDNA genome of 80-180 kb and have been used as biocontrol agents and protein expression vectors. Here, we report on the first synthesis of a baculovirus based on the type species Autographa californica nucleopolyhedrovirus, AcMNPV, by a combination of PCR and transformation-associated recombination in yeast. The synthetic genome, designated AcMNPV-WIV-Syn1, is 145 299 bp comprising the complete genome of AcMNPV except for the hr4a locus that was replaced with an ∼11.5 kb cassette of bacterial and yeast artificial chromosomal elements and an egfp gene. Sf9 insect cells were transfected with AcMNPV-WIV-Syn1 DNA and progeny virus was examined by electron microscopy, and assayed in one-step growth curves and oral infectivity. The results conclusively showed that the rescued virus AcMNPV-WIV-Syn1 had structural and biological properties comparable to the parental virus. We validated a proof of concept that a bona fide baculovirus can be synthesized. The new platform allows manipulation at any or multiple loci and will facilitate future studies such as identifying the minimal baculovirus genome and construction of better expression vectors. This is the largest DNA virus synthesized so far, and its success is likely to be the impetus to stimulate the fields of other large DNA viruses such as herpesviruses and poxviruses.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.|University of Chinese Academy of Sciences , Beijing 100049, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Manli
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Gengfu
authors.initials: G
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.|University of Chinese Academy of Sciences , Beijing 100049, PR China.
authors.forename: Xi
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Dianhai
authors.initials: D
authors.lastname: Hou
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.|University of Chinese Academy of Sciences , Beijing 100049, PR China.
authors.forename: Kai
authors.initials: K
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.|University of Chinese Academy of Sciences , Beijing 100049, PR China.
authors.forename: Shurui
authors.initials: S
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Laboratory for Molecular Virology, Great Lakes Forestry Centre , Sault Sainte Marie, Ontario P6A 2E5, Canada.
authors.forename: Basil M
authors.initials: BM
authors.lastname: Arif
authors.affiliation: Laboratory of Virology, Wageningen University , Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Xinwen
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Hualin
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, PR China.
authors.forename: Zhihong
authors.identifier: 0000-0002-1560-0928
authors.initials: Z
authors.lastname: Hu
mesh_terms: D000818:Animals; D022201:Chromosomes, Artificial / Q000235:genetics; D004267:DNA Viruses / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D016133:Polymerase Chain Reaction; D061987:Sf9 Cells; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D004279:DNA, Viral
keywords: AcMNPV; baculovirus; large DNA virus; synthetic genome