title: Replication-independent assembly of an insect virus (Tetraviridae) in plant cells.
abstract: Infectious virions of the insect RNA virus Helicoverpa armigera stunt virus (HaSV; Omegatetravirus, Tetraviridae) were assembled in cultured plant protoplasts of Nicotiana plumbaginifolia in the absence of detectable replication. Assembly of the virus, which has not been grown in cell culture, required cotransfection of a DNA plasmid expressing the HaSV capsid gene in combination with either genomic RNA or with DNA plasmids carrying the complete cDNAs to the two HaSV genomic RNAs. Each cDNA was placed under the control of the cauliflower mosaic virus 35S promoter and followed by a cis-acting ribozyme so that the resultant transcripts corresponded precisely to the two genomic RNAs. Protoplast assembly of infectious particles was confirmed by EM and bioassay of host insect larvae, which became diseased and produced virus particles confirmed as HaSV. Variant transcripts carrying nonviral sequences at either or both termini of the RNAs showed no infectivity, except for RNA2 carrying only a 3' terminal extension. No replication of HaSV in protoplasts was detected in pulse-labeling and blotting experiments. Insects showed less severe disease symptoms when fed protoplasts transfected with only the RNA1 and coat protein plasmids. The symptomatic larvae contained only RNA1 and failed to yield infectious progeny virus, suggesting that RNA1 is capable of self-replication. This novel plasmid-based system confirms that the reported sequence of HaSV represents an infective genome and establishes a procedure for the reverse genetics of a tetravirus.
authors.affiliation: CSIRO Entomology, Canberra, Australia. karlg@ento.csiro.au
authors.forename: K H
authors.initials: KH
authors.lastname: Gordon
authors.forename: M R
authors.initials: MR
authors.lastname: Williams
authors.forename: J S
authors.initials: JS
authors.lastname: Baker
authors.forename: J M
authors.initials: JM
authors.lastname: Gibson
authors.forename: A L
authors.initials: AL
authors.lastname: Bawden
authors.forename: A G
authors.initials: AG
authors.lastname: Millgate
authors.forename: P J
authors.initials: PJ
authors.lastname: Larkin
authors.forename: T N
authors.initials: TN
authors.lastname: Hanzlik
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D007304:Insect Viruses / Q000235:genetics / Q000502:physiology*; D007814:Larva; D007915:Lepidoptera / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D010947:Plants, Toxic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D014026:Nicotiana / Q000821:virology*; D014162:Transfection; D014779:Virus Replication
chemical_list: D012367:RNA, Viral