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Nucleic Acids Research, 1983, Vol. 11, No. 17 6003-6020
© 1983

MOLECULAR BIOLOGY

Generation of adenovirus by transfection of plasmids

Kathleen L. Berkner and Phillip A. Sharp

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, USA

Received May 20, 1983. Accepted August 8, 1983.

Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plaanids. The covalently attached terminal protein wa3 removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotran3fected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DKA. The above method for preparing adenovirus recomblnants has been used to construct a mutant, Ad5 {bigtriangleup}(78.9–84.3), lacking most of the non-essential EIII transcriptional unit. Thi3 mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.


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