Replication-dependent transactivation of the polyomavirus late promoter

Abstract

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.

Polyomavirus late pre-mRNA processing: DNA replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch

“…This stepwise analysis was necessary because of numerous unusual features of polyomavirus L-mRNAs. The late promoter does not possess a TATA box, which results in the use of at least 15 start sites which span >150 bases (10,13,15,16,(37)(38)(39). Primer extension analysis cannot be used to quantitate leader multiplicity because this technique gives a complex repeating pattern with heterogeneous start sites appearing with a spacing of 57 nucleotides (G. Adami and G. Carmichael, unpublished data).…”

“…The top line represents L-mRNA molecules. The dots represent the major start sites found for RNA produced from the late promoter, with the relative abundances(10,13,15,16,(37)(38)(39) indicated by dot size. The large open rectangle represents the late leader structure, which can contain one or more tandem leader exons.…”

PEA1 and PEA3 enhancer elements are primary components of the polyomavirus late transcription initiator element

“…However, as MT-Ag also activates early transcription initiation (29,56,77,78), it may not directly contribute to a shift in gene expression from early to late transcription initiation during the course of viral infection. LT-Ag may largely contribute to such a shift, probably through multiple mechanisms (8,18,28,67).…”

Cell specificity of transcription regulation by papovavirus T antigens and DNA replication.

“…In rodent cells, PyT and Py DNA replication also lead to an early to late shift in Py mRNA production (Farmerie and Folk, 1984). As in SV40, late Py transcription is activated primarily by the replication process (Cahill et al, 1990). In addition, the processing of Py late transcripts is altered after the appearance of PyT in the cell, thereby contributing to the increased ratio of late to early mRNAs Carmichael, 1988, 1990).…”