PKA/PrKX activity is a modulator of AAV/adenovirus interaction

Abstract

Interference between viruses occurs when infection by one virus results in the inhibition of replication of another virus. Adeno-associated virus (AAV2) is a human parvovirus with the unique characteristics of a dependence upon a helper virus for a productive infection and the ability to interfere with the replication of the helper virus. Previously, we demonstrated that AAV2 Rep78 and Rep52 interact and inhibit cAMP-dependent protein kinase A (PKA) and its novel homolog PrKX. We hypothesized that modulation of PKA activity by AAV2 may be responsible for inhibition of helper virus replication. In this study we demonstrate that adenovirus replication is sensitive to PKA activity and that AAV2 Rep78/Rep52 proteins contain an inhibitory domain similar to that of the heat-stable PKA inhibitor. This domain, while not directly necessary for AAV2 replication and packaging, is necessary to preserve AAV2 replication ®tness during an Ad co-infection. Furthermore, a mutant AAV2 virus lacking this region fails to inhibit adenovirus replication. Thus, inhibition of PKA activity by AAV2 constitutes a novel form of viral interference.

Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

“…When expressed under the control of the authentic promoters in the absence of ITR sequences, only Rep78 and/or Rep52 slightly inhibits adenoviral replication. This inhibition is likely due to the C-terminal domain of Rep78/Rep52, absent in Rep68/Rep40, which was shown to downregulate adenoviral replication by inhibiting cyclic AMP-dependent protein kinase (PKA) through direct protein interaction (31). The dependence of Rep-mediated inhibition on the presence of an amplifiable DNA template also explains the rather weak inhibitory capacity of the Rep proteins under the control of the strong heterologous CMV promoter observed in earlier studies (20).…”

“…The AAV-mediated inhibition of adenoviral replication has been attributed to the expression of the Rep proteins (20,21,31), and based on these findings, the difficulties encountered in various attempts to incorporate the rep gene region into the context of replication-competent first-generation adenoviral vectors (11,12,32) were also assigned to Rep protein expression. A recent study by Sitaraman et al (22) strongly changed this prevailing picture of the rep gene-mediated inhibition of Ad/AAV hybrid vector propagation by showing the involvement of cis inhibitory effects.…”

The Genome of Canarypox Virus

“…Geminivirus Rep proteins affect viral DNA replication and transcriptional repression, induce accumulation of proliferating cell nuclear antigen and bind cellular replication factor C, and bind the cell cycle-regulatory protein retinoblastoma to affect tissue specificity during infection (49,54,66). Human herpesvirus 6 and rat cytomegalovirus contain homologues of parvovirus Rep proteins, endonucleases or helicases which are also multifunctional in affecting viral replication, viral and cellular transcription, site-specific viral integration into host genomes, and interference of helper-virus replication (21,96,100). To our knowledge, CNPV Rep-like proteins are the first to be identified in a poxvirus and could conceivably affect similar functions in CNPV-infected cells.…”

Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

“…AAV inhibits Ad production from severalfold to Ͼ100-fold (7,15,28). Earlier studies implicated CHIP analysis does not necessarily identify DNA-binding proteins since binding and cross-linking can be mediated through other proteins.…”