Characterization of an adenovirus type 5 mutant carrying embedded inverted terminal repeats

Abstract

During construction of an adenovirus type 5 (Ad5) deletion mutant, d1E1,3, lacking E1 and E3 sequences, we isolated a variant, d1E1,3-1, which had a direct repeat of viral DNA terminal sequences attached to the left end of the genome. Analysis of this variant with restriction enzymes and by hybridization of Southern blots with specific probes indicated that the extra terminal segment contained the left 2.6% (920 bp) of Ad5 joined to 352 bp of pBR322 which in turn was linked to the left end (minus 21 bp) of d1E1,3. During replication of d1E1,3-1 the extra terminal segment was found to transfer to the right end of the genome resulting in a second variant, d1E1,3-2, with duplicated terminal sequences at both ends of the viral genome. D1E1,3-2 in turn was shown to revert back to d1E1,3-1 at high frequency. Although evidence was obtained indicating that the extra segment could be lost from the left end, spontaneous mutants which had lost direct repeats from both ends were never isolated. It was, however, possible to remove the extra terminal repeat of d1E1,3-1 by cleavage with a restriction enzyme and to isolate d1E1,3 containing wt termini. The rearrangements occurring during replication of d1E1,3-1 and d1E1,3-2 may be consequences of the mode of replication of Ad5 DNA and bear some resemblance to segment inversion in herpesviruses.