Generation of adenovirus by transfection of plasmids

Abstract

Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was 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 cotransfected 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 DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This 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.