We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177. To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked

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... to the SV40 early or late promoter. Small t increased threeto fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on earlyand late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low. In contrast to the large body of knowledge that exists regarding regulation of cellular and viral gene expression by simian virus 40 (SV40) large T antigen (large T), there are very few experimental data suggesting an explanation for the involvement of small t during productive viral infection. Our observation that SV40 small t can trans activate promoters transcribed by RNA polymerase II (the adenovirus E2A promoter) and III (the adenovirus VA-I promoter) (19) and that polyomavirus small t can also trans activate these promoters (18) suggests the possibility that trans activation may be an essential small t function which has been retained during papovavirus speciation. It is possible that transcription of cellular genes, viral genes, or both occurs in response to small t and that this is important for a successful viral infection. On the other hand, small t is not strictly required for viral infection or transformation in tissue culture. Viruses containing mutations within small t coding regions are not markedly defective but grow slowly (28). SV40-transformed cultured cells can be produced in the absence of small t, and small t in the absence of large T will not transform cells (22) . However, small t appears to be required for transformation when cellular concentrations of large T are low (2, 4). We considered that, similarly, small t may be important only during a certain portion of a viral infection, perhaps when the concentration of large T in cells is low. If this is the case, it may be difficult to observe a requirement for small t during infection of cultured cells if the concentration of large T in cells rises quickly. In previous experiments, trans activation of SV40 early and late genes has been attributed solely to large T (3, 7, 8, 13, 32, 33). However, the involvement of small t, which was also present in these studies, in trans activation of the SV40 * Corresponding author. early and late promoters has not been examined. Large T clearly appears to be required for trans activation of the late promoter, since expression of small t alone is ineffective (19), and some mutations within large T that do not affect small t can completely block trans activation (33). This suggests that certain structures specific to large T are essential for trans activation of the SV40 late promoter. However, it is possible that small t participates with large T in the regulation of the SV40 late promoter. Whether small t alone affects the SV40 early promoter has not previously been examined. In the experiments presented here, we utilized plasmids encoding only large T or small t and found that small t can participate in the regulation of SV40 early and late promoters linked to chloramphenicol acetyltransferase (CAT) plasmids. Possible mechanisms that might explain how small t is involved in transcription regulation are discussed. MATERIALS AND METHODS Cell culture and transfections. CV-1 cells were cultured and transfected with calcium phosphate-coprecipitated DNA as described previously (19, 20) , with the following modifications. Transfections for CAT enzyme and RNA assays were performed with cells cultured in 35-mm plates. Cells were transfected with 2 ,ug of CAT plasmids and, unless otherwise indicated, 2 ,ug of plasmids expressing large T, small t, or mutations. Calf thymus DNA (Clontech) was added to the DNA mixture to bring the total DNA concentration to 15 jig, a DNA concentration which we had determined to be optimal under our transfection conditions. For experiments in which CAT mRNA was to be assayed, six plates were transfected in parallel for each experimental condition. One plate was used for the CAT enzyme assay, and RNA was harvested from the remaining five plates, pooled, and assayed for CAT mRNA. For Western blot 1489 Vol. 66, No. 3 on May 9, 2020 by guest