Covalently closed circles of human adenovirus DNA are infectious.

Abstract

Replication of the linear adenovirus DNA molecule is thought to result from semiconservative synthesis off linear templates, starting from origins at either end of the genome. Recently, however, it has been shown that in cells infected with adenovirus type 5 (Ad5) a significant fraction of the ends of viral DNA molecules become joined head‐to‐tail due at least in part to the formation of covalently closed circles. Circular DNA is not present in virions but joining of the ends of viral DNA is detectable shortly after infection, well before the onset of viral DNA replication. To learn more about the structure and possible function of these circular forms of viral DNA, I have cloned Ad5 circles as plasmids replicating in Escherichia coli. Two plasmids have been analyzed in detail and shown to generate infectious virus with an efficiency comparable with that of virion DNA following transfection into human cells. These results suggest that circles are not totally inert or functionless but that, once formed, they are capable of re‐entering the pool of replicating molecules to generate linear progeny.

Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.

“…Once established, however, these insertion mutants seemed to be quite stable: when a mutant with an insertion of 1.7 kb was passaged five times successively in 293 cells and replaqued, 24 out of 24 plaque isolates analyzed had a structure which was identical to that of the parental mutant (unpublished observations). In other studies we have succeeded in constructing a stable insertion mutant, Ad5in52, with 2.2 kb of inserted DNA (Graham, 1984) but we are not aware of any larger insertions which have been introduced into the AdS genome in the absence of compensatory deletions. Viral and plasmid cloning vectors with deletions of viral DNA To increase the cloning capacity of AdS and thereby extend its utility as a vector, we developed a mutant with extensive deletions in early region 1 (El) and E3 (Haj-Ahmad and .…”

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus

“…The absence of the adenovirus packaging signal from this helper genome completely prevents its packaging into infectious virus particles. The conception of this system was derived from three observations: (1) recombinant baculovirus based on the genome of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) can efficiently transduce certain mammalian cells in tissue culture; [27][28][29] (2) when properly joined, adenovirus ITRs can be efficiently utilized as an adenoviral origin of replication; 30,31 and (3) Cre recombinase can efficiently excise and circularize a DNA fragment flanked by two loxP sites. 32,33 Previous studies demonstrated that AcMNPV is capable of transducing mammalian cells without virus gene expression or replication.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…40 This method was developed based on the construction of an Ad genomic plasmid by inserting pBR322 into the XbaI site in the E1 region of a circularized wt Ad genome isolated from the Ad-infected cell lysate. 41,42 This plasmid, known as pJM17, is too big to be encapsidated because of the insertion of the pBR plasmid sequence. Therefore, it can be used to cotransfect 293 cells with a smaller plasmid (shuttle vector) that contains the left-end arm of the Ad genome (0 -16 mu) with its E1 region substituted by the expression cassette of the transgene.…”