Biophysical evidence for linkage of adenovirus and SV40 DNA's in adenovirus 7-SV40 hybrid particles.

The existence of virus particles in a strain of adenovirus 7 (E46+) consisting of simian virus 40 (SV40) genetic material inside an adenovirus 7 capsid has been demonstrated in several laboratories.'-3 These particles have variously been called "0)," "hybrid,"14 "transcapsidant," or "PARA."5 They are able to induce SV40 T and transplantation antigens, but not SV40 V antigen or virus. IUnderstanding of the possible association of the SV40 DNA with the adenovirus DNA in the "hybrid" particles has been hampered by their defectiveness; they are able to replicate only in cells infected with "nonhybrid" adenovirus and thus cannot be obtained as a pure population free of "nonhybrid" adenovirus. A cell infected with a "hybrid" particle alone yields neither adenovirus nor SV40 progeny. Biologic evidence that the SV40 DNA is linked with Ad. 7 DNA in the "hybrid" was obtained by showing that transfer of the SV40 determinant from one adenovirus type to another regularly resulted in transfer of some genetic material of Ad. 7 into the same particle)7 Nucleic acid homology techniques8 provide a method for specific identification and quantitation of purified DNA's. Ad. 7 complementary RNA (cRNA) prepared in vitro reacts with Ad. 7 DNA but not to any measurable extent with SV40 DNA, and the converse is also true.9 Application of the DNA-cRNA technique to purified E46+ DNA showed that SV40 DNA as well as Ad. 7 DNA was present and that the quantity of each DNA was sufficient to allow biophysical examination of possible DNA-DNA linkage.9 We have used the DNA-cRNA homology technique and a variation of preparative density gradient ultracentrifugation to attempt to separate the SV40 DNA from the adenovirus DNA in the "hybrid" populations under conditions which do separate these DNA's when present in a mixture. Materials and Methods.-Viruses: E46+ virus passed in African green monkey kidney (AGMK) cultures was used as seed material. Pool 1 was prepared by Dr. Maurice Green by infecting KBcell spinner cultures at high multiplicity; pool 2 was grown in AGMK cell monolayers, and pool 3 was grown in KB spinner cultures by Dr. Paul Burnett of the Eli Lilly Co., Indianapolis, Indiana. Human embryonic kidney (HEK) cells were acutely infected with these pools. The ratio of the percentage of these infected cells showing adenovirus T antigen to those showing SV40 T antigen (as measured by fluorescent antibody assay)4 was approximately 2: 1. This is similar to that of all AGMK-grown pools of E46+, and indicates that the "hybrid" and "nonhybrid" particles were present in comparable numbers. E46virus, the "nonhybrid" Ad. 7 recovered from E46 +,2 was grown in KB cells, as was a pool of Gomen strain Ad. 7 virus prepared by Dr. Burnett. This latter pool of Ad. 7 was used as a source of DNA for priming for Ad. 7 cRNA in the second E46+ centrifugation. SV40 virus (strain 777)10 was obtained from Dr. Paul H. Black, and was grown in BSC-1 cell monolayers. All adenovirus preparations were free of adeno-associated viruses (AAV) as judged by electron microscopic examination or complement fixation testing of the preparations against antisera to all four known serotypes.'1 DNA: All DNA preparations were made from virus banded twice in cesium chloride. DNA