The mechanism of internuclear transmission of SV40-induced complement fixation antigen in heterokaryocytes.

During the investigation of the action of SV40 virus by means of fusion of SV40-transformed cells with virus-susceptible monkey kidney cells,' it became apparent that SV40-induced complement fixation antigen (ICFA)2 was transmitted to the nuclei of the nontransformed cells whether or not fusion between the two systems resulted in the isolation of infectious virus.' 3 This was not surprising, since the mechanism of ICFA synthesis apparently is unrelated to the synthesis of viral protein and infectious virus, and the antigen is found in many non-virus-yielding transformed cells. As a corollary of these observations, an investigation of transmission of ICFA in heterokaryocytes to nuclei originating from cells resistant to SV40 infection became of interest. Further, a study of the mechanism of the transmission of ICFA was instituted to elucidate the process of its intracellular synthesis. Materials and Methods.-Tissue cultures: Table 1 lists cell types used for fusion experiments. The origin and history of SV40-transformed human, monkey, and hamster cell lines were described previously.' ICFA (T-antigen)2 was present in cell nuclei of all these lines, referred to as "donor" lines. The "recipient" cells described in Table 1 were characterized by the absence of ICFA in their nuclei. This was confirmed repeatedly during each fusion experiment. All cells were grown in double-strength Eagle's basal medium in Earle's balanced salt solution with 10% fetal calf serum and aureomycin (50 Ag/ml). Technique of cell fusion: The fusion technique was essentially similar to the one described previously.' Recipient cells were labeled with 0.2 uc/ml thymidine methyl-Ha (specific activity 14.5 c/mM) for 4 days prior to fusion. Metabolic inhibitors, FUdR (Hoffman LaRoche, Nutley, N. J.), actinomycin (Lyovac Cosmogen, Merck Sharp and Dohme, Rahway, N. J.), and cycloheximide (Actidione, Upjohn, Kalamazoo, Mich.), were added at the time of fusion, and the fused cells were then grown in inhibitor-containing medium. SV40 ICFA determination and autoradiographic techniques: The presence of ICFA was detected according to the previously described technique.' At least 1000 nuclei were counted under the fluorescent microscope, and nucleograms were constructed, showing the percentage of the total cells counted containing various proportions of fluorescent and nonfluorescent nuclei. The percentage of multinucleated cells containing only ICFA-positive nuclei was determined and used as an index of transmission of ICFA (see Figs. 1, 3, 4, and 5). In some experiments we determined the origin of the nuclei in multinucleated fluorescent cells by subsequent autoradiography of the same cells, as previously described.' Results.-Appearance of ICFA in the nuclei of recipient cells after fusion with transformed ICFA-containing donor cells: The results in Table 1 indicate that ICFA always appeared in the nuclei of the recipient cells after fusion with SV40transformed cells. Transmission of ICFA took place regardless of the origin of the donor and recipient cells and regardless of whether the transformed cell lines were capable of producing infectious SV40 spontaneously or after fusion with