Cellular components interact with adenovirus type 5 minimal DNA packaging domains

Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of a cis-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AT, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respective cis-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting, trans-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.