Studies of adenovirus-SV40 hybrid viruses. V. Evidence for linkage between adenovirus and SV40 genetic materials.

Recent studies have established that the adenovirus type 7 (Ad. 7) strain E46+ is a mixed population of infectious adenovirus particles and defective particles consisting of adenovirus capsids containing the segment of SV40 DNA which codes for T antigen.1-4 Because of the inability to obtain the SV40 DNA carriers as a pure population, it has not been possible to determine whether they also carry adenovirus genetic material and whether there is interaction between the two DNA's at the molecular level. The observation that the SV40 DNA could be transferred from E46+ to other adenovirus types5' 6 provided a technique for testing for such linkage. This report describes transfer of an Ad. 7 gene with the SV40 gene, with evidence that they are present in the same capsid. Materials and Methods.-Viruses: The substrains of the prototype Ad. 2 and Ad. 12 strains which had acquired the SV40 gene by mixed infection with E46+ have been described previously;" they will be referred to here as Ad. 2+(t7) and Ad. 12+(t7) to indicate that they acquired the SV40 gene by transfer from Ad. 7. The pool (pool 1017) of Ad. 2 +(t7) used previously5 was employed for most of the present studies. It represented the tenth African green monkey kidney (AGMK) tissue culture passage after mixture with E46+; passages six through eight were made with Ad. 7 rabbit antiserum, and the ninth was made by limiting dilution passage. The passage history of the Ad. 12 +(t7) virus used here was as follows. Prototype Ad. 12 (Huie) was mixed with E46 + and inoculated into AGMK cultures. The harvest was passed once in AGMK with Ad. 7 rabbit serum, then twice in human embryonic kidney (HEK) cultures with the Ad. 7 antiserum; a plaque was then isolated in AGMIK cells, and this was passed four more times in AGMK. Both virus strains were free of detectable Ad. 7 virus as determined by breakthrough neutralization tests. Plaque isolates were obtained in AGMK cultures, using the procedure described previously.4 Standard rabbit antisera against prototype adenovirus strains were used for neutralization tests. Hamster antisera: The majority of tests for Ad. 7 T antigen were done with a serum pool (pool C) from hamsters carrying transplanted tumors induced by the Pinckney strain of Ad. 7; this strain does not carry the SV40 genetic material. The sera included in this pool were selected for having high-titer (>1:160) complement-fixing (CF) antibody against early cell pack antigens7 of cells infected with Ad. 7. The pool reacted in CF with Ad. 7 tumor extract to a titer of 1: 20, and was negative against standard Ad. 7 viral antigen at 1:10 serum dilution. An additional serum pool and three individual sera from hamsters carrying Ad. 7 (nonhybrid) tumors were used for confirmatory tests; these sera were selected on the basis of reacting in CF with early cell pack antigens of Ad. 7 and/or Ad. 3 and staining intranuclear antigens in cells infected with Ad. 7 in the fluorescent antibody (FA) test. The patterns of immunofluorescent reactivity of the Ad. 7 hamster sera are described below. Pooled serum from hamsters carrying transplanted Ad. 12 tumors was used as a group-reactive immunofluorescent reagent for estimating the total amount of infectious adenovirus.8 Although reactive only with the Ad. 12, 18, 31 subgroup in the CF test, Ad. 12-tumored hamster sera are much more broadly reactive in the FA test. They consistently stain intranuclear antigens in cells infected with Ad. 7 strains,8' 9 but only a few (5 of 16 sera individually tested) stain cells infected with Ad. 2. The serum pool used in the present studies (pool B) stained cells infected with Ad. 1 through 18.