Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system.
cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.
Abrams
C C
CC
BBSRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
King
A M
AM
Belsham
G J
GJ
D005537:Aphthovirus / Q000235:genetics / Q000502:physiology*; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D036022:Capsid Proteins; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant; D000939:Epitopes / Q000032:analysis; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins; D019065:Virus Assembly
D036022:Capsid Proteins; D004274:DNA, Recombinant; D000939:Epitopes; D011498:Protein Precursors; C442501:VP1 protein, Foot-and-mouth disease virus; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus