Immunocytochemistry, autoradiography, in situ hybridization, selective stains: Complementary tools for ultrastructural study of structure‐function relationships in the nucleus. Applications to adenovirus‐infected cells

A significant amount of new information on structure‐function relationships in nuclei of adenovirus‐infected cells has accumulated during the last decade as a result of the combined use of several new cytochemical techniques. Localization of viral DNA on ultrathin sections of infected cells has been investigated at the ultrastructural level by using specific DNA staining and immunocytochemistry with monoclonal anti‐DNA antibodies. Both techniques, however, concomitantly visualize cellular and viral DNA. The specific stain for DNA reveals the configuration of the DNA molecules in the different nuclear substructures, whateer their synthetic activities. The immunodetection of DNA reveals that specific antibodies strongly bind to DNA of condensed host chromatin and to both encapsidated and nonencapsidated inactive viral genomes. However, the observation of an abnormally low level of labeling over the substructures in which synthetic activities of viral genomes are known to be intense demonstrates a serious limitation of this technique for the detection of active DNA. Postembedding in situ hybridization is the most useful method for identifying with certainty the structures containing defined nucleic acid sequences. By using a biotinylated viral DNA probe, in situ hybridization provides specific identification of structures containing either viral DNA or viral RNA molecules. In addition, with appropriate pretreatment of the sections, it is possible to reveal either all the viral DNA‐that is, both double‐ and single‐stranded DNA molecules (dsDNA, ssDNA)–or more specific species such as only ssDNA or only dsDNA molecules. The replicative and transcriptional activities of viral genomes are determined by high‐resolution autoradiography. Autoradiography after a short pulse incorporation of appropriate radioactive precursors by infected cells reveals the sites of cellular and viral DNA replication or trancription. A short pulse followed by chase periods of different durations reveals the progressive migration of the cellular and viral synthesized products. The in situ distribution of the viral 72 kDa DNA‐binding protein, a highly phosphorylated protein which protects the viral ssDNA, is revealed either by immunocytochemistry with specific antibodies or by the bismuth staining method which stains all highly phosphorylated proteins, including both cellular and viral proteins. The combined results of all these cytochemical procedures reveal the composition and functions of some of the structures induced by adenovirus infection. They demonstrate that viral genomes engaged in replication lead to the formation of replicative foci in which two compartments rapidly develop, one of which results from the aggregation of single strands of viral DNA and their accompanying 72 kDa protein. Conversely, ssDNA and 72 kDa protein are rare in the other compartment which is the main site of replication and transcription of viral genomes. The procedural aspects and the contributions of electron microscope cytochemistry to an understanding of the biology of Ad5 viruses can serve as a basic framework for the study of other biological systems. © 1995 Wiley‐Liss, Inc.