Bovine papillomavirus transcriptional regulation: localization of the E2-responsive elements of the long control region

Abstract

The long control region (LCR) of the bovine papillomavirus type 1 genome can function as a conditional transcriptional enhancer which can be specifically trans-activated by the viral E2 gene product. To precisely map the target(s) of this trans-activation, BAL 31 exonuclease was used to generate two overlapping series of deleted DNA segments through the LCR. These fragments were assayed for their ability to activate transcription from the enhancer-deleted simian virus 40 early promoter of pA10CAT in the presence or absence of the viral E2 gene product. Two different E2-responsive elements were localized within the LCR. The major target for E2 trans-activation (E2-responsive element 1) was mapped to a 196-base-pair fragment between nucleotides 7611 and 7806, just upstream from promoters P7940 and P89. Further deletions which destroyed or impaired enhancer function revealed that the ACCN6GGT sequence motifs at each end of E2-responsive element 1 are critical components of this element. Primer extension analysis of RNA extracted from acute transfections with plasmids containing the bovine papillomavirus type 1 LCR driving the CAT gene revealed that each of the P7940 and P89 promoters is responsive to E2 trans-activation.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

“…In this paper we use this assay to define for the first time the 449 two viral factors that together are necessary and sufficient for viral DNA replication. One of these factors is the viral transactivator E2, which appears to be required in a capacity other than the hitherto reported role as a regulator of BPV transcription (Spalholz et al, 1985;Spalholz et al 1987;Lambert et al, 1987;Stenlund and Botchan, 1990). The other factor is a 72 kd polypeptide that is encoded by the El open reading frame in its entirety.…”

Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.

“…After selection in The respective mutants in this fragment are depicted and are described further in Materials and methods. The following genetic elements are indicated: NCOR-1 and 2, negative control of replication 1 and 2; PMS-1, plasmid maintenance sequence 1; ORI, minimal origin of replication; E2RE1 and E2RE2, E2-responsive enhancer 1 and 2 (Spalholz et al, 1987) the long-term replication assay, however, the result was very different. While the plasmid containing the 2.5 kb BglII fragment could be readily detected in episomal form, the MO-containing plasmid could not be recovered (compare lanes 12 and 13, Figure 2B).…”

The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.

“…Reporter plasmids 3FSVCAT and 6FSVCAT were constructed by insertion of three (3F) or six (6F) copies of the consensus p53 binding sequence TGCCT upstream of the enhancer-deleted SV40 promoter in the CAT reporter plasmid pAlOCATBS (Spalholz et al, 1987). Doublestranded oligonucleotides prepared from the following sequences: 5'-GATCTCCTTGCCTGGACTTGCCTGGCCTTGCCTTTTG-3' (3F) and 5'-GATCTCCTTGCCTGGACTTGCCTGGCCTTGCCTTCCT TGCCTGGACTTGCCTGGCCTGCCTTTG-3' (6F) were used to insert the p53 target sequences between the BglII and SalI sites of pAlOCATBS.…”