Specificity of enzyme-substrate interactions in foot-and-mouth disease virus polyprotein processing.
A series of transcripts derived from FMDV cDNA plasmids containing defined regions of the genome were translated in a rabbit reticulocyte lysate system. The products were analysed directly or following incubation with an FMDV-infected cell processing extract. Processing by the L proteinase at the L/1A cleavage site occurred when most of the P1-2A protein was absent. Substitution of sequences upstream of the 2C/3A cleavage site showed that the 3C proteinase was also able to cleave at an entirely novel cleavage site, apparently at K-I amino acid pairs. Cleavage at the 2A/2B site was not only independent of L and 3C proteinases, but was shown to occur when 2A and as few as four 2B N-terminal amino acids were present. Thus, the disparate proteolytic activities responsible for all three primary processing events that give rise to the products L, P1-2A, 2BC, and P3 were highly resistant either to major deletion or substitution of protein sequences adjacent to, or at, the site of cleavage. By contrast, secondary processing in trans was sensitive to changes at remote sites. For example, removal of the C-terminal regions of P1-2A and 2BC precursors impaired their ability to act as substrates for 3C proteinase activity. Processing of P1-2A, particularly of the 1D/2A cleavage site, was enhanced by inclusion of sequences from the 3D region of the genome.
Ryan
M D
MD
A.F.R.C. Institute of Animal Health, Pirbright Laboratory, Woking, England.
Belsham
G J
GJ
King
A M
AM
D005537:Aphthovirus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D011498:Protein Precursors; D014764:Viral Proteins; D010450:Endopeptidases