The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment.

BACKGROUND: Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data. RESULTS: The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations. CONCLUSIONS: The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid.

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.

Structure determination of feline panleukopenia virus empty particles.

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 380.1 A, b = 379.3 A, and c = 350.9 A, has been determined to 3.3 A resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 A resolution and gradually extended to 3.3 A resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA.

Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus.

Analysis of canine parvovirus (CPV) isolates with a panel of monoclonal antibodies showed that after 1986, most viruses isolated from dogs in many parts of the United States differed antigenically from the viruses isolated prior to that date. The new antigenic type (designated CPV type 2b) has largely replaced the previous antigenic type (CPV type 2a) among virus isolates from the United States. This represents the second occurrence of a new antigenic type of this DNA virus since its emergence in 1978, as the original CPV type (CPV type 2) had previously been replaced between 1979 and 1981 by the CPV type 2a strain. DNA sequence comparisons showed that CPV types 2b and 2a differed by as few as two nonsynonymous (amino acid-changing) nucleotide substitutions in the VP-1 and VP-2 capsid protein genes. One mutation, resulting in an Asn-Asp difference at residue 426 in the VP-2 sequence, was shown by comparison with a neutralization-escape mutant selected with a non-CPV type 2b-reactive monoclonal antibody to determine the antigenic change. The mutation selected by that monoclonal antibody, a His-Tyr difference in VP-2 amino acid 222, was immediately adjacent to residue 426 in the three-dimensional structure of the CPV capsid. The CPV type 2b isolates are phylogenetically closely related to the CPV type 2a isolates and are probably derived from a common ancestor. Phylogenetic analysis showed a progressive evolution away from the original CPV type. This pattern of viral evolution appears most similar to that seen in some influenza A viruses.