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.

Plant protoplast technology: Current status

“…Pol len proto plasts have im proved our un der stand ing of wall de vel op ment in re la tion to plant re pro duc tion (Zhao et al 2004). As a re sult of their abil ity to take up macromolecules, proto plasts have fea tured as ex per i men tal ma te rial to im prove un der stand ing of plant-vi rus in ter ac tions, in clud ing as sem bly of an in sect vi rus in plant cells (to bacco; N. plumbaginifolia) (Gordon et al 2001). Other work ers have also emphasised the rel e vance of proto plasts in virus stud ies (Choi et al 2003).…”

Subcellular localization and live-cell imaging of the Helicoverpa armigera stunt virus replicase in mammalian and Spodoptera frugiperda cells

“…Research into tetravirus biology has therefore been limited to the heterologous expression of viral proteins in E. coli (Zhou et al, 2006(Zhou et al, , 2008, Saccharomyces cerevisiae (Tomasicchio et al, 2007), insect tissue-culture cell lines (Agrawal & Johnson, 1995) or transfected plant protoplasts (Gordon et al, 2001).…”

Effective Control of a Field Population of <I>Helicoverpa armigera</I> by Using the Small RNA Virus <I>Helicoverpa armigera</I> Stunt Virus (<I>Tetraviridae</I>: <I>Omegatetravirus</I>)

“…A new approach, however, is made possible by biotechnology and termed "nonhost production" . The feasibility of this approach demonstrated by Gordon et al (2001) where HaSV was assembled in cells that did not support its active replication. A scale-up of this process in industrial organisms such as yeast or plants could bring to bear an effective, cheaply produced weapon against the worldÕs most economically important group of insect pests.…”