Adeno-associated virus Rep78 protein interacts with protein kinase a and its homolog PRKX and inhibits CREB-dependent transcriptional activation

Adeno-associated virus (AAV) is a human parvovirus of the genus Dependovirus. AAV replication is largely restricted to cells which are coinfected with a helper virus. In the absence of a helper virus, the AAV genome can integrate into a specific chromosomal site where it remains latent until reactivated by superinfection of the host cell with an appropriate helper virus. Replication functions of AAV have been mapped to the Rep68 and Rep78 gene products. Rep proteins demonstrate DNA binding, endonuclease, and helicase activities and are involved in regulation of transcription from both AAV and heterologous promoters. AAV has been associated with suppression of oncogenicity in a range of viral and nonviral tumors. In this study we sought to identify and study cellular protein targets of AAV Rep, in order to develop a better understanding of the various activities of Rep. We used the yeast two-hybrid system to identify HeLa cell proteins that interact with AAV type 2 Rep78, We isolated several strongly interacting clones which were subsequently identified as PRIM (previously named PKX1), a recently described homolog of the protein kinase A (PKA) catalytic subunit (PKA,). The interaction was confirmed in vitro by using pMal-Rep pull-down assays. The region of Rep78 which interacts was mapped to a C-terminal zinc finger-like domain; Rep68, which lacks this domain, did not interact with PRIM. PRIM demonstrated autophosphorylation and kinase activity towards histone H1 and a PKA oligopeptide target. Autophosphorylation was inhibited by interaction with Rep78. In transfection assays, a PRIM expression vector was shown to be capable of activating CREB-dependent transcription. This activation was suppressed by Rep78 but not by Rep68. Since PRIM is a close homolog of PKA,, we investigated whether Rep78 could interact directly with PKA,. pMal-Rep78 was found to associate with purified PKA, and inhibited its kinase activity. Cotransfection experiments demonstrated that Rep78 could block the activation of CREB by a PKA, expression vector. These experiments suggest that AAV may perturb normal cyclic AMP response pathways in infected cells.