A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein.
Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.
Brown
L M
LM
Department of Biological Sciences, University of Western Sydney, Macarthur, P.O. Box 555, Campbelltown, NSW 2560, Australia. louise.brown@agric.nsw.gov.au
Papa
R A
RA
Frost
M J
MJ
Mackintosh
S G
SG
Gu
X
X
Dixon
R J
RJ
Shannon
A D
AD
D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006041:Goats; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011995:Recombination, Genetic; D012697:Serine Endopeptidases; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004279:DNA, Viral; D018985:Epitopes, B-Lymphocyte; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases