ASSEMBLY OF VIRUS-LIKE PARTICLES IN INSECT CELLS INFECTED WITH A BACULOVIRUS CONTAINING A MODIFIED COAT PROTEIN GENE OF POTATO LEAFROLL LUTEOVIRUS

DNA encoding the coat protein (P3) of a Scottish isolate of potato lea froll virus (PLRV) was inserted into the genome of Autographa californ ica nucleopolyhedrovirus (AcNPV) such that the coat protein was expres sed either in an unmodified form or with the addition of the amino aci d sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H), Insect cells i nfected with these recombinant baculoviruses accumulated substantial a mounts of P3 and P3-6H. P3 could not be recovered from cell extracts u nless it was denatured in SDS but a proportion of the P3-6H was recove rable in a soluble form in nondenaturing conditions, Immunogold labell ing of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies, In contrast, although much of the P3-6H al so accumulated in nuclei, it formed virus-like particles (VLP) which w ere often grouped in close-packed, almost crystalline arrays, When ele ctron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistingu ishable from PLRV particles trapped from extracts of PLRV-infected pla nts, The VLP co-sed imented in sucrose gradients with PLRV particles w hich suggests that the VLP contained RNA, VLP collected from sucrose d ensity gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a prote in, with an M(r) of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the co at protein gene, This protein was also found in the nuclear fraction o f infected cells but was more readily soluble than was P3.