1.13.10
INSECT CELLS INFECTED WITH RECOMBINANT BACULOVIRUSES PRODUCE PARTICLES THAT RESEMBLE VIRIONS OF POTATO LEAFROLL VIRUS

MA MAYO1, B REAVY1, GH DUNCAN1, FE GILDOW2, JAT WOODFORD1 and JW LAMB3

1Scottish Crop Research Institute, Dundee, UK; 2Department of Plant Pathology, Pennsylvania State University, State College, PA, USA; 3Biochemistry Department, University of St Andrews, UK

Background and objectives
In previous work, when the coat protein gene of potato leafroll virus (PLRV) was expressed in insect cells from a recombinant baculovirus, virus-like particles (VLP) were obtained only if the protein carried extra N-terminal amino acids [1]. We have prepared another recombinant baculovirus that contains a full-length cDNA copy of the PLRV genome, and co-infected cells with this and the previous construct in an attempt to recover functional virions.

Materials and methods
This construct containing the modified PLRV coat protein gene was as described by Lamb et al. [1]. The full-length construct was prepared from cDNA clones reverse-transcribed from PLRV RNA and by mutagenesis to obtain a terminal sequence corresponding to that of PLRV. The cDNA was transferred via pVL1392 into baculovirus DNA, where it was cloned behind the polyhedrin gene promoter. Insect cells were inoculated with preparations of recombinant baculoviruses and VLP were recovered from them between 2 and 3 days after inoculation.

Results and conclusions
Buffer extracts of baculovirus-infected cells and thin sections of cells contained particles that resembled those of PLRV. Northern blot analysis showed that PLRV-specific RNA of about 5-6 kb was produced in the infected cells and that the VLP contained this RNA. Protein analysis showed that the VLP did not contain protein P5.

VLP sedimented in sucrose gradients at a rate similar to that of PLRV particles. However, the VLP were not infective when inoculated to isolated tobacco protoplasts or when supplied to aphids by injection or by membrane feeding. Nevertheless, VLP were observed in the aphid bodies, including in the lumen of the salivary gland duct [2]. Lack of P5 in the VLP seems not to prevent passage of the particles through the aphid body.

References
1. Lamb JW, Duncan GH, Reavy B et al., 1996. Journal of General Virology 77, 1349-1358.
2. Gildow FE, Reavy B, Mayo MA et al., 1997. Phytopathology 87, S33.