Construction of adenovirus expression vectors by site-directed in vivo recombination.

We developed a method for conveniently positioning foreign DNA at many preselected sites in the adenoviral genome by a combination of in vitro and in vivo recombination. Using this technique, we constructed a set of recombinant viruses that contain the SV40 A gene downstream from the adenovirus tripartite leader. One of these hybrid viruses, Ad-SVR26, contains the A gene close to and downstream from both the major late promoter and the first segment of the tripartite leader. The transcripts encoded by the inserted SV40 DNA are highly overproduced in infected cells; they initiate at the adenoviral late promoter and terminate at the SV40 polyadenylation site. Several novel splice acceptor sites in the SV40 sequences are used in the processing of the primary transcript to produce six different species of spliced RNA. The synthesis of T antigen in Ad-SVR26-infected cells requires the use of novel AUG initiation codons present within the SV40 coding region or adenoviral sequences that normally form part of the intron between the first and second segments of the tripartite leader. The level of T antigen expression is not as high as the level of mRNA production. The usage of these new AUG triplets or the absence of the complete adenovirus tripartite leader sequence may account for the low efficiency of translation.