Immunolocalization of vaccinia virus structural proteins during virion formation

Proteolytic processing of vaccinia virus core proteins is an essential step in the formation of mature virions and occurs during the process of virion morphogenesis. In order to investigate how the vaccinia virus (VV) structural proteins become integrated into virus particles during normal maturation, immunological reagents were generated against the three major VV core proteins 4a, 4b, and 25K and their precursor molecules P4a, P4b, and P25K. These sera were used in conjunction with immunofluorescent and immunogold labeling of VV-infected tissue culture cells. The immunofluorescent results indicated that all three core precursors and their cleavage products were localized to virosomes. As the infection progressed, punctate staining with these sera became spread throughout the cytoplasm which suggested that individual virion particles were being recognized. Immunoelectron microscopy showed that the core proteins were localized to the center of both immature and mature virus particles. This result was in contrast to the situation observed using antisera directed against L65, a protein previously implicated in the assembly of the viral membrane. Immunogold staining of L65 showed that it was initially located along the inner side of the immature virion membrane and remained with the membrane even as the viroplasm began to condense toward the center of the virus particle. In order to determine whether the core protein localization observed was the result of precursors, products, or both, a synthetic peptide strategy was used to generate an antiserum that recognized only P4a in immunoprecipitation reactions. Immunogold labeling with this reagent indicated that P4a was found in the viroplasm of immature particles and in low levels in the mature virion. Intracellular localization of core and L65 proteins during virion morphogenesis is discussed.