Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors.
Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor P1-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins 1AB, 1C and 1D were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike P1 processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting L-containing cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-P1 junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.
Roosien
J
J
Department of Virology, Agricultural University Wageningen, The Netherlands.
Belsham
G J
GJ
Ryan
M D
MD
King
A M
AM
Vlak
J M
JM
D000818:Animals; D005537:Aphthovirus / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D004274:DNA, Recombinant; D004279:DNA, Viral; D010447:Peptide Hydrolases