Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.

Direct intratumoral (i.t.) injection of adenoviruses (Ads) expressing specific immunostimulatory cytokines represents an attractive strategy for the clinical implementation of cytokine gene therapy of cancer. Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells and promotes a T helper 1-like immune response. We have constructed an Ad vector (AdCMV-mIL-12) containing both chains of the murine IL-12 (mIL-12) gene linked by an internal ribosomal entry site sequence under the transcriptional control of the cytomegalovirus immediate-early gene promoter, which is able to mediate the transient expression of very high levels of biologically active mIL-12 both in vitro and in vivo. An i.t. injection of 4 ϫ 10 8 plaque-forming units of AdCMV-mIL-12 resulted in a complete regression of day 7 established subcutaneous MC38 murine adenocarcinomas and MCA205 murine fibrosarcomas. Treated animals rejected a subsequent rechallenge with MC38 and MCA205, respectively, demonstrating the induction of long-lasting antitumor immunity. Specific antitumor cytotoxic T lymphocyte reactivity was detected in splenocytes harvested from treated animals. A significant increase in the numbers of both CD4 ϩ and CD8 ϩ T cells in the AdCMV-mIL-12-infected tumors was observed. Ad-mediated IL-12 gene therapy was also associated with measurable serum levels of mIL-12 and profound changes in the composition of splenic lymphocytes. Taken together, these results demonstrate the feasibility and efficacy of delivering IL-12 directly i.t. using a recombinant adenoviral vector.

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase a-binding, and transcriptionalautoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specffic and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.