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 leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density 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 protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
Lamb
J W
JW
School of Biological and Medical Sciences, University of St Andrews, UK.
Duncan
G H
GH
Reavy
B
B
Gildow
F E
FE
Mayo
M A
MA
Hay
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004279:DNA, Viral; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine; D017793:Luteovirus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000821:virology; D018411:Spodoptera / Q000166:cytology; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure; D019065:Virus Assembly
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D006639:Histidine