Pulse labeling of cells with [(35)S]methionine or [(3)H]glucosamine at different times after infection, followed by SDS-PAGE and Western immunoblotting analysis using rabbit anti-TCV hyperimmune serum, was used to resolve and identify TCV-induced intracellular proteins. The viral structural proteins (gp200, gp140/gp66, gp100/gp120, p52, and gp24/p20) were detected in radiolabeled cell extracts by 9 to 12 hours post-infection, as well as two possible non-structural proteins with apparent mol.wts. of 36,000 and 32,000. The predominant 52,000 nucleocapsid protein could be detected in cell lysates as soon as 6 to 8 hours after infection; it was initially resolved as a complex of 3 closely migrating species with mol.wts. ranging from 46,000 to 52,000. Pulse-chase and immunoprecipitation experiments indicated that gp200 arose from a putative precursor with mol.wt. of 150,000 to 170,000, that underwent glycosylation. Proteolytic cleavage of gp200, in turn, probably yielded the gp100 and gp120 species. The unique TCV hemagglutinin protein originated from a primary precursor with mol.wt. of 60,000, which underwent rapid dimerization by disulfide bond formation and glycosylation to yield gp140. The peplomeric and matrix proteins were both shown to be N-glycosylated, as indicated by their sensitivity to tunicamycin (TM) and their resistance to sodium monensin (SM). In the presence of TM, proteins with mol.wts. of 90,000, 120–130,000, and 150,000 accumulated in TCV-infected cells rather than peplomeric glycoproteins, and the matrix protein E1 was only detected in its unglycosylated form. The addition of TM to the culture medium interfered with the maturation of progeny viral particles, as suggested by the absence of peplomers at the surface of the intravacuolar and extracellular virions, and the accumulation of amorphous material not found in the absence of the glycosylation inhibitor. High yields of virus replication were obtained, in the presence of SM, even at concentrations which greatly affected the cellular functions.
Intracellular synthesis and processing of the structural glycoproteins of turkey enteric coronavirus