Expression of AAV Rep Proteins in SV40-Transformed and Untransformed Cells: Reciprocal Interaction with Host DNA Synthesis

Abstract

Adeno-associated virus (AAV) inhibits the induction of host DNA synthesis by simian virus 40 (SV40) large-tumour (T) antigen, mediated through AAV-encoded ‘Rep’ regulatory proteins. Rep proteins are normally synthesized by AAV-infected cells only in the presence of adenovirus. However, we observed a low level of Rep protein expression in SV40 transformed cells even in the absence of helper virus. In an effort to understand the functional interaction between SV40 T antigen and regulators of AAV rep expression, we evaluated Rep protein production by cell lines transformed with various T antigen mutants known to vary in their induction of host DNA synthesis. We observed Rep protein expression proportional to SV40-induced host DNA synthesis, as measured previously for these T antigen mutants in the absence of AAV, suggesting that rep gene expression – although it opposes the oncogenic stimulation of cell cycling by SV40 – may itself be elicited by host DNA synthesis. To test this, we employed two inhibitors of DNA synthesis: hydroxyurea, which acts by depleting deoxyribose nucleotide triphosphate pools, and aphidicolin, a specific inhibitor of DNA polymerases α and δ. Each inhibitor markedly and significantly reduced Rep protein levels, both in immortal cells transformed by wild-type T antigen and in normal human fibroblasts, confirming the dependence of Rep protein expression on host DNA synthesis.