Identification of domains in canine parvovirus VP2 essential for the assembly of virus-like particles

Abstract

Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VP1. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.

Molecular characterization of canine parvovirus variants (CPV-2a, CPV-2b, and CPV-2c) based on the VP2 gene in affected domestic dogs in Ecuador

“…The EC/C3 (CPV-2) was used to construct the 3D view of the VP2 protein and the location of all substitutions of the four complete sequences is located in each segment. Among the aa substitutions of sample 1 in residues within Loop 1-4 already reported by other researchers [ 7 , 33 ], the additional substitution in residue 514 appears out of the most antigenic region of the protein [ 47 ], not making clear the possible effect that this may have on the antigenic function of the viral capsid. Substitution in residue 514 is shown in Figure-4 , showing no more structural changes than a loss of a molecule of oxygen.…”

Antigen-Capture Blocking Enzyme-Linked Immunosorbent Assay Based on a Baculovirus Recombinant Antigen to Differentiate Transmissible Gastroenteritis Virus from Porcine Respiratory Coronavirus Antibodies

“…The cells were grown in suspension or monolayer culture as previously described. 17 Autographa californica multiple nucleopolyhedrovirus (AcMNPV) wild type e was used to purify DNA for the transfections based on the p10 promoter. Recombinant baculoviruses AcYM1gX-SA, AcYM1gX-SB, AcYM1gX-SC, and AcAS3gX-SB-His, which express the TGEV S protein-fragment A, B, or C under polyhedrin or p10 gene promoters, were used for antigen preparation.…”

Virus‐Like Particles: Models for Assembly Studies and Foreign Epitope Carriers

“…The specific three-dimensional structure of the Canine or Porcine parvovirus (CPV and PPV, respectively) VP2, with its four loops between eight-stranded antiparallel b-barrel motifs, appears to be a particularly suitable site for the insertion of epitopes to produce VLPs as carriers of molecules for antigen delivery. Two regions of VP2 dispensable for capsid formation, the N terminus which is directed toward the inside of the VLP, and loop 2, which is partially presented on the surface of the capsid, proved suitable sites for foreign epitope insertion in antigen presentation when expressed in insect cells (136)(137)(138)(139). To elucidate events related to CPV infection, fluorescent VLPs were developed.…”