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

Human adenovirus type 5 (Ad5) contains a 36‐kb double‐stranded DNA molecule in an icosahedral capsid. Attempts to construct Ad5 insertion mutants containing DNA of more than about 105% of the genome size resulted in viral progeny in which deletions had occurred suggesting the existence of severe constraints on the size of packageable DNA molecules. To partially circumvent these constraints we used an adenovirus vector, Ad5dlE1,3, with deletions in early regions 1 (E1) and 3 for a total net reduction in genome size of 5349 bp and an expected capacity for inserts of greater than 7 kb. To use this vector efficiently we generated a circular form of dlE1,3 DNA which could be propagated as an infectious bacterial plasmid. When this plasmid was used as a recipient for inserts of various sizes it was found that its capacity was much less than expected and that dlE1,3 virion capsids could not even package DNA as large as the wt genome. Because the E1 deletion of dlE1,3 extends into the coding sequences for protein IX, a minor capsid component known to affect the heat stability of adenovirions, the possibility that absence of this polypeptide might also affect the DNA capacity of the virion was investigated. It was found that when the coding sequences for protein IX were restored the packaging capacity of the vector was also restored to that of wt virions. Thus protein IX is an essential constituent of virion capsids dispensable only for virions containing DNA of less than genomic size.