Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells
Journal of Virology
The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Spl, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus
... nd reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Spl promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Spl or bound by other activator proteins does not restore growth. This indicates that Spl or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Spl activates both SV40 transcription and DNA replication. TX 77030. which viral transcription and DNA replication might be modulated (2, 95, 99). Little or nothing is known about how most of these proteins affect SV40 gene function or replication. We have attempted to resolve their importance by substituting synthetic sequences capable of binding individual proteins in place of the 21-bp repeat region and then measuring their effects upon virus growth in CV-1 cells. Our results indicate that two high-affinity Spl motifs serve nearly as well as the intact 21-bp repeat region in activating SV40 early gene expression and, concomitantly, DNA replication and plaque formation. In contrast, binding sites for several other protein activators do not substitute for the 21-bp repeat region. Because much is known about Spl structure and function, we speculate about how Spl binding to the 21-bp repeat region might promote both DNA replication and DNA transcription. MATERIALS AND METHODS Plasmid constructions. Standard procedures were employed in cloning experiments (86) with Escherichia coli XL1-Blue (Stratagene, La Jolla, Calif.). The construction of key plasmids is described below. (i) pCV21-0. Since the 21-bp repeat region is not flanked by unique restriction enzyme sites, we devised pCV21-0 to facilitate subsequent constructions (Fig. 1B) . Cloning vector pCV21-0 contains the SV40 genome (776 strain) with the 21-bp repeat region replaced with prokaryotic filler DNA. The DNA fragments used to construct pCV21-0 were derived from pX113 and pS312 (29) and pBRX, a derivative of pBR322 in which the SspI site was converted into an XhoI site by the insertion of an 8-bp XhoI linker. The gap that results from the removal of the filler DNA (after double digestion with SalI andXhAol) is referred to as the cloning site of pCV21-0. (ii) pWTSV40. pWTSV40 contains the 776 strain of SV40 cloned at EcoRI into the same vector as pCV21-0. (iii) p21-0. p21-0 was constructed by removing the filler DNA in pCV21-0, which yielded the sequence shown in Fig. 6379 on May 10, 2020 by guest http://jvi.asm.org/ Downloaded from sequence identifies nt 34. Spl motifs are boxed. Sall (S) and XhoI (X) sites are underlined.