Although the specificity of the ribonuclease and the findings of the control experiments permit these results to stand on their own, it was thought advisable to attack the question from an additional and unrelated angle. While the electron microscope preparations were being made, other samples of the virus-containing regions of Rous pellets were smeared on glass slides and allowed to dry. The smears were then fixed and stained with acridine-orange for nucleic acids, the staining being done either directly, or after incubation for 2 hours at 37° C. in ribonuclease, or after similar incubation in control medium without the enzyme.

When structures containing ribonucleic acid stained with acridine-orange are examined in ultraviolet light, they usually exhibit an orange-red fluorescence, which specifically identifies the presence of ribonucleic acid if it is abolished by ribonuclease digestion before the staining (17, 18). Although light microscopy obviously cannot resolve individual Rous virus particles, electron micrographs of sectioned pellets had established that the material used in the acridine-orange staining experiments consisted almost wholly of the virus (fig. 4). Thus, the over-all fluorescence response obtained with and without ribonuclease digestion can be taken as a reflection of the response of the individual particles to the acridine-orange staining.

The presence of much ribonucleic acid in the Rous virus was therefore confirmed by the intense orange-red fluorescence of the virus when stained directly, by the absence of such fluorescence when stained after ribonuclease digestion, and by the retention of the orange-red fluorescence in the control preparations which had been treated with enzyme-free medium alone (16).

To digress briefly, it is interesting to note that this type of integration of electron microscopy with cytochemical methods is not something which can be applied only to the Rous virus and its ribonucleic acid. It has wider applications, both in the tumor virus field and in the field of virology in general, as has been shown by a recent similar study of an infectious virus containing nucleic acid of the deoxyribose type (19).

The experiments just described give good evidence that another tumor virus, in addition to the virus of avian myeloblastosis, namely, the Rous virus, contains ribonucleic acid and this is supported by the reported failure to detect any deoxyribonucleic acid in biologically active though relatively crude preparations of this agent (20).

Recently information has been obtained regarding the type of nucleic acid probably present in a mammalian tumor virus quite unrelated to the avian agents so far considered. DiMayorca et al. (21) succeeded in making an infectious protein-free extract of polyoma-infected tissue-culture cells and showed that the infectivity was susceptible to treatment with deoxyribonuclease. This suggests that the polyoma virus, the presumed source of the infectivity, contains nucleic acid of the deoxyribose type; for more conclusive evidence the nucleic acid would have to be extracted from purified virus rather than from virus-infected cells. However, were suitable purified virus preparations available, other methods, such as those used for the avian tumor agents, could be usefully applied.

Leaving the practical considerations, the information available regarding tumor virus composition, though scanty, raises important questions concerning the basic biological nature of such agents and the possible manner in which they bring about malignant change.

It is sometimes suggested that tumor viruses have quite a special relationship with their host cells and, in the case of the Rous virus, at any rate, speculation has been current regarding some kind of genetic interaction. It is difficult, however, to see how this could take place unless widely accepted views of the primary genetic mechanism operating in cells are to be discarded, since the Rous agent is of the ribonucleic acid type. Admittedly, it is not known whether ribonucleic acid has any ultimate control of genetic pathways, but if, as knowledge increases, a mode of interaction for tumor viruses with this kind of nucleic acid appears more feasible, the phenomenon could hardly be genetic according to the present understanding of the word, unless the units of cellular heredity were also found to contain ribo- as well as deoxyribonucleic acid. At the present time there is no evidence whatsoever in the field of tumor virology to suggest that such genetic interaction between virus and host cell takes place (22). Explanations as to how such an interaction might hypothetically occur in the case of the ribonucleic acid-containing Rous virus must therefore be regarded as speculation in support of speculation.

There are besides this two other theoretical ways in which tumor viruses might produce oncogenic effects, and neither of these calls for the revision of accepted principles of cell biology. In either case one must assume that tumor viruses and ordinary infective viruses behave essentially in a similar manner when they parasitize cells and replicate. The existence of such a similarity is supported by the findings on tumor virus composition reviewed previously. For, the polyoma virus provides an example of a tumor agent that probably relies on deoxyribonucleic acid for its genetic continuity, whilst the Rous and myloblastosis viruses contain ribonucleic acid, thus showing that tumor viruses can possess nucleic acid of either type just as can ordinary viruses.

The manner in which these two groups of agents differ must lie merely in the rate of their replication and its resultant pathogenicity for the cell; it could then be that the tumor viruses act as such simply because during their relatively slow replication process they cause some sublethal damage to the cell which leads to its unfettered multiplication. According to one view this might be by the destruction of the ability to register the presence of outside stimuli which normally act as a check on the systems serving cell division, these systems then being allowed free rein. According to the other theory, the process of replicating a tumor virus could so pervert and damage the finely balanced normal interactions within the cell, that some enzyme system might be released from control or actually stimulated and thus bring about unlimited cell division where such should not occur.