JOURNAL OF VIROLOGY, Jan. 1990, p. 347-353 Vol. 64, No. 1 0022-538X/90/010347-07$02.00/0 Copyright C 1990, American Society for Microbiology Programmed Factor Binding to Simian Virus 40 GC-Box Replication and Transcription Control Sequences ROBERT L. BUCHANANt AND JAY D. GRALLA* Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90024 Received 6 July 1989/Accepted 22 September 1989 Nuclear footprinting revealed a temporal program involving factor binding to the repetitive GC-box DNA elements present in the simian virus 40 regulatory region. This program specified ordered and directional binding to these tandem regulatory sequences in vivo during the late phase of infection. The program was interrupted by the DNA replication inhibitor aphidicolin or by inactivation of the viral replication factor simian virus 40 T antigen, suggesting a link between viral DNA replication and new factor binding. Measurements of DNA accumulation in viruses lacking either the distal or proximal halves of the GC-box region suggested that the region has a dual role in replication control. Overall, the data point to important relationships between DNA replication and factor binding to the GC-box DNA, a multifunctional regulatory region. Factor binding to mammalian regulatory DNA elements is MATERIALS AND METHODS of central importance in gene control. The cellular distribu- Viral infections, preparation of nuclei, and DNA purifica- tion and activity of such factors likely contributes to cell- tion. SV40 strain tsA58 and parental strain VA45-54 were specific patterns of gene expression. Although many DNA- obtained from Peter Tegtmeyer. In general, CV-1 cells were binding factors have been identified (see reference 26 for an infected with 1 to 5 PFU of SV40 per cell and maintained in example), the mechanisms that control their distribution and Dulbecco modified Eagle medium plus 2% calf serum plus activity are not understood. Many models relevant to such antibiotics. Enough cells were infected to provide approxi- mechanisms center on programmed factor binding to DNA mately 0.1 to 0.5 ,ug of SV40 DNA per footprinting reaction. and especially on the influence of DNA replication on such Isotonic nuclei lysed with Nonidet P-40 (Sigma Chemical programs (see, for example, reference 5). Co.) were prepared and nicked with DNase I as previously Study of mammalian DNA tumor viruses has revealed described (6). DNA was purified as previously described (6), well-defined genetic programs that depend directly or indi- except that the order of RNase A and proteinase K diges- rectly on DNA replication (45). In simian virus 40 (SV40) tions was reversed to use endogenous RNA as a carrier in and adenovirus, components of the replication and transcrip- DNA precipitations. tion machinery involved in such programs have been purified In some experiments, DNA replication was inhibited (14, 26). Most of the protein components are host cell before isolation of infected nuclei. At 32 h postinfection, the proteins, which suggests that regulation of viral programs cell culture medium was brought to 5 ,ug of aphidicolin has important features in common with cellular regulation. (Sigma) per ml and infections were continued for up to 16 h Among these proteins are cell factors which control SV40 (29). replication and transcription through common DNA ele- Primer extension and gel electrophoresis. The sites of ments (10). A critical DNA target of these cellular proteins is DNase I nicking were determined by hybridizing 5'-end32P- the repeated motif (GGGCGG)6 or the GC box which is a labeled oligonucleotide primers to denatured samples and regulatory element adjacent to the viral core origin of extending them to breaks with Klenow DNA polymerase replication that influences DNA replication (24, 31, 32), as essentially as previously described (6, 20), except that all well as early (1, 11) and late transcription (4, 13). The role of extension reactions were done at 50 to 52°C. Base denatur- these factors in uninfected cells is also likely to be involved ation of viral DNA templates gave a lower background than with gene regulation, since GC boxes are often associated heat denaturation for samples isolated before 40 h. Base with regulatory DNA (27, 38). denaturation and subsequent primer extension have already In situ DNase I footprinting of nuclear SV40 templates been reported (3). isolated from infected CV-1 monkey cells showed abundant Quantitation of replicated viral DNA. Confluent CV-1 cells factor binding to the SV40 GC boxes at 48 h postinfection, were infected with equal titers of wild-type SV40 mutant but neither the identity ofthe factors nor their functional role virus strain 776, SV-P7, or PXS7. After 2 h, infected plates is known (6). In this report, we identify a genetic program were washed three or four times with warm Dulbecco involving this factor and present experiments suggesting that modified Eagle medium to reduce the amount of unattached the programmed binding requires templates competent to virus. Viral DNA was prepared as described above and replicate. Since GC boxes and repetitive DNA are relatively electrophoresed on 1% agarose gels after linearization with common in cells, these observations may have interesting EcoRI. Full-length nick-translated ([32P]dATP) FIII SV40 implications for programming interactions with cellular reg- DNA was used to probe DNA from 12- to 24-h postinfection ulatory DNA. samples by Southern hybridization. DNA that hybridized to the labeled SV40 probe was visualized by autoradiography * Corresponding author. with Cronex film at room temperature. Several autoradio- t Present address: Division of Biology 156-29, California Institute grams at different exposures were scanned by densitometry. of Technology, Pasadena, CA 91125. Viral DNA samples prepared from experiments done at 24 347 348 BUCHANAN AND GRALLA J. VIROL. 5183 5203 5223 5243/0 20 40 60 80 100 120 I I EARLYmRNA 4- ILAE mRNA .- * L 21 21 21 T AG 1 TAG 2 T AG3 * 5145 -* 5230 11 III IV v v I AATT AGTCAGCCAT GGGGCGGAGA ATGGGCGGAA CTGGGCGGAG TTAGGGGCGG GATGGGCGGA GTTAGGGGCG GGACTATGGTT 28 31 41 51 61 71 81 91 101 FIG. 1. The SV40 control region with oligonucleotide primers used in footprinting experiments. The bidirectional arrow designates the linear SV40 DNA sequence with nucleotide numbers according to Tooze (45). The directions of early and late transcription are indicated. Relevant SV40 control regions are also shown, including the centers of three T-antigen (T AG)-binding domains. Three arrows labeled 21 depict the six GC-box regulatory elements. The wedge labeled 72 is the origin-proximal portion of the 72-base-pair viral enhancer element. Oligonucleotide primers are shown as arrows with numbers (5145 and 5230) indicating the 3' ends of the primers. At the bottom, the SV40 GC-box region is expanded to show the nucleotide sequence from nts 28 to 111. Within this expanded region, the GC boxes are numbered with roman numerals. ORI, Origin of replication. to 65 h postinfection were linearized and electrophoresed on was partially protected from DNase I digestion by a bound 1% agarose gels after dilutions typically in the range of 1:10 factor at 40 h postinfection (6). The technique detects only to 1:1,000. DNA was visualized by ethidium bromide stain- the interaction of abundant factors with nonencapsulated ing and photographed with Kodak Tri-X film. The amount of DNA; the approximately 90% of SV40 DNA in virions and DNA in each lane was determined by densitometry. A range previrions is relatively DNase I resistant (see reference 6) of exposures were taken to assure linear response. and does not contribute significantly to the signal in this assay. Factor binding to less than half of the remaining 10% RESULTS of the DNA is difficult to detect with this method (6). The late phase of the SV40 life cycle involves programmed Probing the replication core region in vivo. Figure 2 shows production of viral DNA and protein with the goal of the results of in situ probing of the replication core se- assembling infectious virus (45). As the late phase proceeds, quences and the adjacent T-antigen-binding site I. Factor is assessed of DNA com- more RNA is produced from the expanding pool of DNA, binding by comparing digestion with 2 to but the average transcriptional activity of SV40 DNA tem- plexed protein (nuclei, lanes 4) with digestion of plates remains constant (18). However, the average replica- naked DNA (in vitro, lane 1). There was significant protec- tion activity of SV40 DNA decreases during this time (24, tion of sequences over T-antigen site I at 32, 40, and 48 h 33). Thus, instead ofthe exponential production of DNA that postinfection. However, strong protection of sequences cor- would accompany uncontrolled replication, the amount of responding to the core elements for replication which are SV40 DNA simply increases linearly. This relationship be- adjacent to this site was not observed (compare lanes 2 to 4 tween replication and transcription apparently ensures a with lane 1). balanced ratio of protein to DNA. The purpose of these These footprinting results are in excellent accord with initial experiments was to probe the in situ interactions with long-standing observations in the literature. At this time in DNA elements near and within the replication core during the lytic cycle, the dominant interaction in this region is the time when the programmed decrease in average template expected to involve T antigen bound at site I acting as a activity occurs. repressor of early transcription (34). Immunological studies The method of probing DNA-protein interactions (6, 20) indicated that approximately 10% of SV40 DNA in vivo is involves adding DNase I to nuclei isolated from SV40- bound by T antigen (42), and this agrees well with strong infected CV-1 cells to lightly nick the endogenous SV40 protection of approximately 10% of the templates in these DNA.