Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.
The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.
Bloom
M E
ME
Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Disease, Hamilton, Montana 59840, USA. mbloom@nih.gov
Martin
D A
DA
Oie
K L
KL
Huhtanen
M E
ME
Costello
F
F
Wolfinbarger
J B
JB
Hayes
S F
SF
Agbandje-McKenna
M
M
D000454:Aleutian Mink Disease Virus / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D002460:Cell Line; D000939:Epitopes / Q000276:immunology*; D015870:Gene Expression; D008907:Mink; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; C442555:VP2 protein, Aleutian mink disease virus