Three-Dimensional reconstructions of ’light’ and ’intermediate’ capsids of equine herpes virus

Abstract

Equine herpes virus type 1 (EHV-1) belongs to an extensive family of large, genetically complex, and medically important animal viruses. The virion consists of an icosahedral nucleocapsid (T=16) separated from the viral envelope by a proteinaceous tegument layer. Assembly occurs in the nucleus of infected cells where capsids assemble, are packaged with DNA, then bud through the nuclear membrane. Two morphological species of EHV-1 capsids have been distinguished: “lights” which are abortive particles incapable of packaging DNA and “intermediates” which are precursors in the assembly of mature virions. Purified “intermediates” contain an additional proteinP22 (46 kDa), which is not present in “lights” and accounts for ˜10% of the total particle mass. In order to characterize the capsid structures of the two particle types and explore differences between them, we have applied three-dimensional reconstruction techniques to electron micrographs of frozen-hydrated specimens.The Kentucky A strain of EHV-1 was propagated in L-929 cells, and the two types of capsids were obtained as separate fractions from Renografin-76 density gradients. Neither fraction contained any significant amount of DNA. Cryo-electron microscopy of capsids on carbon film substrates was performed as described earlier. Micrographs were recorded at a nominal magnification of x36,000 at 2-3μm underfocus and digitized with a 50/μn step size (˜1.38nm sampling at the specimen). Three-dimensional reconstructions of the two particle types were computed to 4.5nm resolution using modified “common-lines” procedures. Images of 37 “light” and 39 “intermediate” particles were separately combined to compute three-dimensional density distributions.