Recombinant Adenoviruses (Ads) have been shown to have potential
applications in three areas: gene therapy, high level protein expression and
recombinant vaccines.' At least three different locations within the Ad
genome can be deleted and subsequently used for the insertion of foreign
sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4)
regions. Viral vectors of this type have been well studied in Human Ads 2
and 5, however one has not yet been constructed for Bovine Adenovirus
Type 2 (BAV2).
The E3 region is located between 76.6 and 86 m.u. on the r-strand
and is transcribed in a rightward direction. The gene products of the Early
3 region (E3) have been shown to be non-essential for viral replication, in
vitro, but are required for host immunosurveillance. This study
represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A
deletion of 1800bp was made within the E3 region of BAV2 and the
thymidine kinase gene was subsequently inserted in the deleted area . . The
plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate
homologous recombination with the wild type BAV2 to produce a mutant.
Southern Blotting and Hybridization results suggest the presence of a
BAV2 E3 deletion mutant with thymidine kinase sequences present.
The E4 region of Human Adenovirus types 2 and 5 is located at the
extreme right end of the genome (91.3 map units - 99.1 map units) and is
transcribed in a leftward direction giving rise to a complicated set of
differentially spliced mRNAs. Essentially there are 7 open reading frames
(ORFs) encoding for at least 7 polypeptides. The gene products encoded by
the E4 region have been shown to be essential for the expression of late
viral genes, host cell shutoff and normal viral growth. We have cloned and
sequenced the right end segment between 90.5 map units and 100 map
units of the BAV2 genome. The results show several open reading frames
which encode polypeptides exhibiting homology to three polypeptides
encoded by the E4 region of human adenovirus type 2. These include the
14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and
the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction
enzyme map, and ORF map of the E4 region could be very useful in future
molecular manipulation of this region and could possibly explain the slow
growth rate of BAV2 in MDBK cells