Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function

Abstract

The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.

Molecular Biology, Epidemiology, and Pathogenesis of Progressive Multifocal Leukoencephalopathy, the JC Virus-Induced Demyelinating Disease of the Human Brain

“…In order to avoid misunderstanding, the second convention is less confusing, as four NFI genes exist, NFI-A, -B, -C, and -X (alternatively called -D) (176). The two identical sites in the 98-bp palindromic repeat element (NFI II/III) were shown to be more important for early gene transcription than the unique third element on the late side of the NCCR (NFI I) (260,455). These sites were also shown to be more important for late gene expression (259).…”

“…Mad-1 NCCR transcription factor binding sites include four Oct-6/tst-1/SCIP binding sites (528), two Pur␣ binding sites (79), two YB-1 binding sites (79,232), two LCP-1 binding sites (479), two GF-1 binding sites (78), four NFI binding sites (13,14,455,483), and six Spi-B binding sites (314). The Mad-1 NCCR is composed of two 98-bp tandem repeats, each containing a TATA box (Fig.…”

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

“…The sites of mutation were finally confirmed by DNA sequencing by the dideoxy chain termination method by using USB sequencing kits as described earlier [26].…”

“…To determine the promoter activity, we utilized the bacterial chloramphenicol acetyl transferase (CAT) assays as described previously [26]. Briefly, subconfluent monolayers of NIH3T3 fibroblast cells were transfected with 10 Ìg of the reporter plasmid DNA by the calcium phosphate precipitation method.…”

“…Site-specific mutants were constructed by the method following Kunkels as described previously [26]. The mismatched oligonucleotide primers were synthesized chemically as follows and the site of mismatch is underlined:…”