IDENTIFICATION OF THE MAJOR MEMBRANE AND CORE PROTEINS OF VACCINIA VIRUS BY 2-DIMENSIONAL ELECTROPHORESIS

Vaccinia virus assembly has been well studied at the ultrastructural l evel, but little is known about the molecular events that occur during that process. Towards this goal we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV p reparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane protei ns. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and S-35 labeling, were identified by either matrix-as sisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixt een major spots that partitioned into the NP-40-DTT-soluble fraction w ere identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described c ore proteins, two of which were also detected in the membrane fraction . Subsequently, the NP-40-DTT-soluble and -insoluble fractions from pu rified virus preparations, separated by 2D gels, were compared with po stnuclear supernatants of infected cells that had been metabolically l abeled at late times (6 to 8 h) postinfection. This relatively short l abeling period as well as the apparent shutoff of host protein synthes is allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequen tly treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified th e major late membrane and nonmembrane proteins of the IMV as they occu r in the virus as well as in infected cells. This 2D gel map should pr ovide an important reference for future molecular studies of vaccinia virus morphogenesis.