Replication-independent assembly of an insect virus (Tetraviridae) in plant cells

Infectious virions of the insect RNA virus Helicoverpa armigera stunt virus (HaSV; Omega tetra virus, Tetraviridae) were assembled in cultured plant p rotoplasts of Nicotiana plumbaginifolia in the absence of detectable replic ation, Assembly of the virus, which has not been grown in cell culture, req uired cotransfection of a DNA plasmid expressing the HaSV capsid gene in co mbination with either genomic RNA or with DNA plasmids carrying the complet e cDNAs to the two HaSV genomic RNAs. Each cDNA was placed under the contro l of the cauliflower mosaic virus 35S promoter and followed by a cis-acting ribozyme so that the resultant transcripts corresponded precisely to the t wo genomic RNAs. Protoplast assembly of infectious particles was confirmed by EM and bioassay of host insect larvae, which became diseased and produce d virus particles confirmed as HaSV. Variant transcripts carrying nonviral sequences at either or both termini of the RNAs showed no infectivity, exce pt for RNA2 carrying only a 3' terminal extension. No replication of HaSV i n protoplasts was detected in pulse-labeling and blotting experiments. Inse cts showed less severe disease symptoms when fed protoplasts transfected wi th only the RNA1 and coat protein plasmids. The symptomatic larvae containe d only RNA1 and failed to yield infectious progeny virus, suggesting that R NA1 is capable of self-replication, This novel plasmid-based system confirm s that the reported sequence of HaSV represents an infective genome and est ablishes a procedure for the reverse genetics of a tetravirus. (C) 2001 Aca demic Press.