The A Enhancer of Polyomavirus: Protein-Protein Interactions for the Differential Early and Late Promoter Function under Nonreplicating Conditions

Abstract

The A enhancer of the polyomavirus early promoter is a 110-bp domain located in the late region and it contains the major late RNA initiation site. The ‘core’ of this enhancer binds several cellular proteins, including proteins PEA1, PEA2 and PEA3. Another element, NF-D/YY1, is also located in this enhancer. The A enhancer is known to stimulate the early promoter, contains auxiliary elements for replication and serves as the initiator for late transcription. It may also be involved in early-to-late switch. We were interested in investigating how the A-core- and NF-D-binding proteins regulate early and late promoter activity under nonreplicating conditions and how the protein-protein interactions affect the function of the individual elements. By point mutational analysis, we show that, except for PEA1, all other proteins activate the early and late promoters differentially under nonreplicating conditions. All three core-binding proteins, and the protein bound to the NF-D site, are activators and have a combinatorial effect on early promoter activity. On late transcription, only PEA1 acts positively and inactivation of the NF-D site is without any effect. In contrast, PEA2 and PEA3 have a repressor-like activity under nonreplicating conditions, indicating that these two proteins might be involved in repressing late transcription, probably early in infection. By increasing the spacing between two consecutive elements, we further show that protein-protein interaction is important for enhancer function. Transactivation of the early promoter was affected by mutations in all four protein-binding sites. Responsiveness of these factors in regard to the late promoter was parallel to their intrinsic promoter strength. The effects of middle and large T antigens are parallel for both the early and the late promoter, suggesting that the pathway(s) for transactivation function of these oncoproteins may overlap downstream. This study with cloned viral promoter will be reflective of situations in vivo, at least partially, under nonreplicating conditions.

Natural Biology of Polyomavirus Middle T Antigen

“…Binding sites in the ␣ core portion of the enhancer (24 bp) include those for PEA1, PEA2, and PEA3 (236). In addition, a binding site(s) for NF-D (nuclear factor D)/YY1 (yin and yang 1) is located outside the core of the ␣ enhancer (51,218,314). At least two of PEA1, PEA3, and PEA2 are required for Py DNA replication, although the specifics, as outlined below, are still unclear.…”

Inhibition of Polyomavirus ori- Dependent DNA Replication by mSin3B

“…c-Ets-1 can abolish c-Jun-stimulated transcription in a construct containing the PEA3/AP1 element of the Py enhancer (27). PEA2 and PEA3 have repressor-like activities on late transcription under nonreplicating conditions (76). Py tumor antigens also may repress early gene expression at late times (20) and late gene expression at early times (9).…”