Histochemical Studies of Lysosomes and Lysosomal Enzymes in Virus-infected Cell Cultures

The appearance of lysosomes and the distribution of lysosomal enzymes have been studied in a number of cell cultures exposed to viruses. Lysosomes were shown by fluorescence microscopy after vital staining with aminoacridines and light microscopy after vital staining with neutral red. The lysosomal enzymes studied histochemically in unfixed and fixed cells were acid phosphatase and 5-bromo-4-chloro-indoxyl acetate esterase. Activation of lysosomal enzymes was found to take place in three stages. The first is characterized by permeability of lysosomal membranes without release of enzymes. This is demonstrable by staining of lysosomal enzymes in unfixed cells and by increased uptake of aminoacridine fluorochromes and neutral red into lysosomes. In cell sheets initially stained with neutral red this gives rise to red plaques. This stage can be fully reversible; cells infected with, and yielding, the red-plaque strain of NDV, recover fully afterwards. In the second stage lysosomal enzymes are released into the cytoplasm, the cells round up and there is decreased uptake of aminoacridines and neutral red into lysosomes. In cell monolayers this results in the formation of white plaques. In the third stage, not usually seen in cell cultures, lysosomal enzymes are released from or inactivated in the cells and are not seen in either fixed or unfixed preparations. The possible roles of lysosomal enzymes in production of cytopathic effects, polykaryocytosis and malignant cell transformation are discussed.