Characterization of adenovirus-induced inverted terminal repeat-independent amplification of integrated adeno-associated virus rep-cap sequences

Stable packaging cell lines expressing the rep and cap genes for recombinan t adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa rep-cap cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the i nverted terminal repeats (ITRs) deleted. We now report a more detailed anal ysis of this phenomenon and highlight the key cellular and viral factors in volved. Determination of the rep-cap copy number of HeRC32 cells indicated that maximum rep-cap amplification occurred between 24 and 48 h following a denovirus infection. Analysis by pulsed-field gel electrophoresis of adenov irus-infected HeRC32 cells indicated that amplified rep-cap sequences were found in an extrachromosomal form. Amplification of the rep-cap sequence wi th the ITRs deleted was not dependent on adenovirus replication and still o ccurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indic ating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was esse ntial, since rep-cap amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant enc oding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of rep-cap amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing t he DBP also simultaneously expressed the Rep proteins, suggesting a possibl e involvement of the latter in rep-cap amplification. Indeed, the lack of d etectable amplification in an adenovirus-infected stable rep-cap HeLa cell clone unable to produce Rep proteins further supported that, among the vira l gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated rep-cap sequences in the absence o f the AAV ITRs.