| The Adeno-associated viruses (AAVs) are single-stranded DNA (ssDNA)- containing parvoviruses under development for therapeutic gene delivery applications. However, a need to improve vector production requires a better understanding of their capsid assembly and genome packaging mechanisms. The parvoviruses are reported to assemble their empty capsids in the nucleus prior to genome packaging by the Rep proteins. However, it still remains unclear exactly how these processes occur. The Rep proteins encoded by the genome have been shown to interact with the AAV capsid proteins and the ssDNA genome, but Rep-capsid protein complexes have not previously been visualized and the region on the capsid surface at which Rep interacts has not been structurally mapped. We have applied biochemical, molecular, and structural approaches to attempt identification of the Rep binding site on the surface of the AAV capsid using wt AAV2 and an AAV2 packaging deficient mutant, AAV2-R432A, shown to have an increased affinity for Rep proteins. A FLAG-tagged AAV2-R432A capsid was constructed to enrich the yield of the Rp-capsid complexes, and putative packaging complexes were observed by negative stain electron microscopy. However, immunolabeling experiments of these Rep-capsid complexes with gold particles were not conclusive in pinpointing the globular attachment as Rep. Cryo-electron microscopy and image reconstruction of wild-type and AAV2-R432A capsids at low resolution have indicated that a closure of the 5-fold pore may be responsible for the inability of AAVR432A to package viral genome. Further structural rearrangements observed in the AAV2-R432A capsid may lead to disruption of intermonomer interactions, and ultimately may serve to weaken the capsid, as was confirmed by DSC studies. These findings provide better insight into the mechanism of AAV genome packaging. |
