| Adeno-associated virus (AAV) is gaining momentum as a vehicle for delivering genes and DNA material to neuronal tissues. However, there remain impediments to the development of the virus as research and gene therapy tools. One impediment is the difficulty of producing the virus in large amounts and high purity. We developed a protocol for isolating AAV from producer cells that takes advantage of the virus' nuclear assembly. The protocol resulted in recovery of 7-fold more recombinant AAV from producer cells than if the virus was recovered through detergent lysis of producer cells and resulted in AAV that contained far fewer contaminants than was obtainable with other methods. Another impediment in the development of AAV technology is the incomplete understanding of the biology of the virus, including its nuclear targeting by the capsid proteins during initial infection and during progeny viral assembly. We demonstrate that the AAV putative nuclear localization signal, 166PARKRLN 172, is not necessary for trafficking the capsid proteins into the nucleus during viral assembly, but the region is necessary for the initial infection by AAV virions. We show that mutation of the 166PARKRLN 172 region does not interfere with trafficking of AAV capsids or genomes to the nuclear periphery, but virions containing the mutation are unable to facilitate transfer of their encapsulated DNA into the nucleoplasm. Virions missing VP2 were fully infectious, demonstrating that the 166PARKRLN 172 region from VP2 is not required for infectivity. We show that another basic region in the AAV capsid, 307RPKRLN 311, plays a role in concentrating AAV capsid proteins in the nucleus during assembly of progeny virions. We mutated several kinase consensus sequences near the AAV 166PARKRLN172 region to determine whether the possible phosphorylation sites are important in the infectivity of the virus. We mutated each of three possible kinase phosphorylation targets to either alanine or aspartic acid. We found one site, serine 181, for which transduction efficiency was enhanced by the S181A substitution and for which transduction efficiency was abrogated by the converse S181D substitution. Taken together, the data presented here make strides in AAV production technology and in understanding the molecular biology of the virus. |
