Simultaneous detection of highly phosphorylated proteins and viral major DNA binding protein distribution in nuclei of adenovirus type 5-infected HeLa cells.

Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism.