Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1).

Three distinct species of nucleocapsids of equine herpesvirus type-1 (EHV-1) were isolated from infected L-M cell nuclei. The particles were classified on the basis of their densities in Renografin gradients as Light (L; ? = 1.237 g/cc), Intermediate (I; ? = 1.244 g/cc), and Heavy (H; ? = 1.258 g/cc). Analysis of the three nucleocapsid species, radioactively labeled with an ³H-labeled or ¹⁴C-labeled amino acid mixture, by discontinuous SDS-polyacrylamide-gel electrophoresis revealed significant and reproducible differences in their structural protein compositions. H nucleocapsids were comprised of six major proteins (I, II, III, IV, IVa, and V) with respective molecular weights of 148,000, 59,000, 46,000, 37,000, 30,000, and 18,000; these proteins comprised 63.7, 9.3, 6.0, 10.8, 5.3, and 4.1%, respectively, of total protein. These six proteins were also present in I nucleocapsids, however nucleocapsid protein IVa (MW, 30,000) was present only as a minor component and comprised less than 1% of total protein. L nucleocapsids were comprised of only four of these major structural proteins, as proteins III (MW, 46,000) and IVa (MW, 30,000) were absent. In addition, several minor proteins, each comprising less than 1% of total nucleocapsid protein, were present in each nucleocapsid species.Electrophoretic analysis of nucleocapsids labeled with [³H]arginine indicated that proteins extremely rich in arginine are not present in these three species. Phosphoproteins both of enveloped virions and of nucleocapsids were identified by electrophoretic analysis of particles radioactively labeled with inorganic phosphate (H₃³²PO₄). Twelve virion structural proteins were phosphorylated in vivo; five of these, including the major virion phosphoprotein VP 10 (MW, 127,000), were envelope specific proteins. Four of these 12 viral proteins were also phosphorylated in nucleocapsids, however the pattern of phosphorylation of nucleocapsid proteins varied among the three species. The two major phosphoproteins of nucleocapsids were proteins III and IVa which are absent in L nucleocapsids; protein V is phosphorylated only in H nucleocapsids.Analysis of the DNA content of the three nucleocapsid species indicated that preparations of H nucleocapsids contain more DNA than do those of the I and L species. Electron microscopic analysis of the nucleocapsids supported these results, as L and I nucleocapsids lack a dense inner nucleoid structure characteristic of the H species.