Effects of Adeno-associated Virus on Adenovirus Replication and Cell Viability
Abstract (Summary)Adeno-associated virus (AAV) is a non-pathogenic parvovirus that requires adenovirus (Ad) or another helper virus for efficient viral replication. During coinfections, AAV inhibits Ad replication and gene expression and induces numerous alterations in the cell. Although these effects are well-documented, many details of these interactions are unknown. In Manuscript One, we examined the effects of AAV on Ad replication and gene expression during coinfections. Coinfection with AAV reduced Ad production by as much as fifty-fold in terms of infectious virions, and forty-fold in terms of DNA replication. With the exception of the E3 gene, mRNAs generated from all Ad transcription units decreased in a dose-dependent response to AAV, but not all transcription units were affected equally. Ad protein expression roughly paralleled mRNA levels, which suggests that AAV does not have major effects on translation during coinfections. We also examined the relationship between reduced E2A and E4 gene expression and decreased Ad DNA synthesis. Expression of Rep78 protein following plasmid transfection did not inhibit E2A and E4 expression prior to DNA replication, and AAV-specific reductions in E2A and E4 transcript levels did not occur in the presence of hydroxyurea. This suggests that decreasing Ad early gene expression is not the primary mechanism by which AAV decreases Ad DNA replication. In Manuscript Two, we examined the effects of AAV on cell viability and expression of the E3 Adenovirus Death Protein (ADP). This was accomplished using Ad5 and an Ad5- derived ADP mutant designated pm534. Lack of ADP expression eliminated cytolysis during the first five to six days of infection without affecting AAV helper functions. During coinfections with Ad5, AAV decreased ADP protein levels. Cell viability was also reduced after day 4, but it was increased prior to that time. Based on studies conducted with AAV/pm534 coinfected cells, the early increase in cell lysis is likely due to AAV-induced apoptosis, which is characterized by Annexin-V staining, ZVADdependent inhibition of apoptosis, caspase activation, and changes in morphology that are characteristic of apoptotic cells. We also observed alterations in extracellular pH that may be related to AAV-induced apoptosis. These data provide the first haracterization of AAV-induced apoptosis in the context of a viral infection.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:adenovirus adeno associated virus apoptosis
Date of Publication:01/01/2006