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We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.