Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein.
The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the endosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function normally. However, an anti-c-Myc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64.
Zhou
Jian
J
Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, NY 14853, USA.
Blissard
Gary W
GW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000914:Antibodies, Viral; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D016259:Genes, myc; D006863:Hydrogen-Ion Concentration; D009036:Moths / Q000821:virology; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity / Q000502:physiology; D011487:Protein Conformation; D017510:Protein Folding; D018411:Spodoptera; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D004279:DNA, Viral; D000939:Epitopes; D015678:Viral Structural Proteins