We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes. © 2011 Alba et al.