Expression of AAV Rep proteins in SV40-transformed and untransformed cells: reciprocal interaction with host DNA synthesis.
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 alpha and delta. 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.
Batchu
R B
RB
Myeloma and Transplantation Research Center, University of Arkansas for Medical Sciences, Little Rock, Ark 72205, USA.
Shammas
M A
MA
Wang
J Y
JY
Shmookler Reis
R J
RJ
Munshi
N C
NC
D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000378:metabolism; D016590:Aphidicolin / Q000494:pharmacology; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006378:Helper Viruses / Q000235:genetics / Q000502:physiology; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D000957:Antigens, Viral, Tumor; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D016590:Aphidicolin; D007041:Hypoxanthine Phosphoribosyltransferase; D004265:DNA Helicases; D006918:Hydroxyurea