Binding of an SV40 T antigen-related protein to the DNA of SV40 regulatory mutants

Abstract

When simian virus 40 (SV40) infects host cells, the viral genes are expressed in two phases. Early in infection, the virus genome is transcribed and translated to yield two related proteins, ‘large T’ and ‘small t’ antigens. Later the rate of synthesis of the early messenger RNA falls and the viral genes coding for the coat proteins are transcribed and translated. Early gene transcription is inhibited only when an active large T antigen is present in the cell (for review see ref. 1). These and other observations have led to the hypothesis that T antigen acts as a negative feedback repressor of its own RNA transcript^2–4. The properties of SV40 tsA mutants, which produce temperature-sensitive T antigen, provide evidence for another role of T antigen—in the initiation of viral DNA replication⁵. Mutations around the origin of DNA replication in SV40 lead to a cis-acting defect in DNA replication^6,7. These mutations fall outside the region of the A gene which codes for T antigen but within the region known to bind T antigen⁸ and the closely related and more easily prepared D2T protein^9,10 which is synthesized by an adeno–SV40 hybrid virus Ad2D2 and seems to differ from SV40 T antigen only in its amino-terminal segment¹¹. Revertants of origin-defective mutants have been isolated with second site mutations within the DNA A gene coding sequence¹². These data support the view that T antigen modulates transcription and replication of SV40 DNA by binding to one or more sites in and around the viral origin of replication. We present here evidence to show that mutants of SV40 carrying constructed mutations in the regulatory region¹³ show reduced binding of D2T protein in vitro. This is the first demonstration in a eukaryote that a mutation in a regulatory DNA sequence alters the binding of a controlling protein in vitro.