Characterization of cis-Acting Sequences Involved in Canine Adenovirus Packaging

Abstract

The cis-acting packaging domain in adenovirus serotype 5 (Ad5) is a series of redundant, albeit not functionally equivalent, “A-repeats” made up of the consensus sequence 5′-TTTGN8CG-3′. A-repeats may bind trans-acting factors that direct packaging of the adenovirus genome into the preformed capsid. To try to understand this basic mechanism, we examined the packaging domain from a nonhuman adenovirus. We delimited the canine adenovirus type 2 (CAV-2) packaging domain to within 156 bp via a conditional mutation based on the Cre/loxP excision. Using an insertion, deletion, and substitution strategy, we generated packaging-defective CAV-2 vectors. Our results demonstrate that, like Ad5, CAV-2 cis-acting packaging sequences are located near the left inverted terminal repeat and are redundant, but not functionally equivalent. However, the bipartite motif found in Ad5 is present only once in CAV-2 and deletion of it caused only a minor variation in the packaging efficiency. We have identified at least four functional cis-acting packaging sequences in CAV-2. The CAV-2 vectors that we generated were not replication-defective in an E1-transcomplementing cell line and as heat stable as the parental vectors that did not contain mutations.