Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins.

Abstract

The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyhransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.

Alleviation of Human Papillomavirus E2-Mediated Transcriptional Repression via Formation of a TATA Binding Protein (or TFIID)-TFIIB-RNA Polymerase II-TFIIF Preinitiation Complex

“…Moreover, since the inclusion of constitutive enhancer elements I and II (CEI and CEII [ Fig. 2A]), which are functional in vivo (13,28), does not enhance E6 promoter activity (Fig. 2D, compare lanes 1 and 11 and lanes 6 and 16), this result also suggests that the HPV-11 enhancer elements are not active in vitro when transcriptional assays are performed on naked DNA templates with HeLa nuclear extracts.…”

“…Plasmids p7862-70GLess/I ϩ and p7862-70GLess/I Ϫ containing a truncated HPV-11 URR from nucleotide 7862 to nucleotide 70 without CEI and CEII were similarly linked to G-less cassettes with or without the MLP Inr. The compilation of cis-acting elements and trans-acting factors in the HPV-11 URR is mainly based on published information (28,44,72). The boundaries of CEI (28) and CEII (13) and the origin of replication (7) cassette templates, which can be used for mechanistic studies of HPV transcription (see below).…”

“…The compilation of cis-acting elements and trans-acting factors in the HPV-11 URR is mainly based on published information (28,44,72). The boundaries of CEI (28) and CEII (13) and the origin of replication (7) cassette templates, which can be used for mechanistic studies of HPV transcription (see below). General cofactors, such as TAF II s and PC4, and phosphorylation on E2 are not required for E2-mediated repression of the E6 promoter.…”

Control of HPV 18 DNA replication by cellular and viral transcription factors

“…The BPV1 E2 can either activate or repress papillomavirus promoters (14)(15)(16). In genital HPVs, the only demonstrated transcriptional regulation by E2 is the repression of the viral early promoters (17)(18)(19)(20)(21), although it can also activate transcription when bound upstream of heterologous promoters. The specific DNA binding by E2 is essential for papillomaviruses DNA replication in vivo (22).…”

Differentiation-independent Constitutive Expression of the Human Papillomavirus Type 16 E6 and E7 Oncogenes in the CaSki Cervical Tumour Cell Line

“…) are strongly implicated in the pathogenesis of cervical cancer (Beaudenon et al, 1986;Boshart et al, 1984;Broker, 1987;Di Luca et al, 1986;Durst et al, 1983;Pirisi et al, 1987;Woodworth et al, 1989;zur Hausen, 1977). The HPV genome is approximately 8 kb of DNA including two reading frames encoding the E6 and E7 oncogenes (Broker, 1987;Hirochika et al, 1988;Seedorf et al, 1985). Expression of E6 and E7 is sufficient for immortalization of cultured epidermal keratinocytes (Munger et al, 1989) and the activity of the P97 promoter, which regulates E6/E7 production, is regulated by both HPV and cellular proteins (Chan et al, 1989(Chan et al, , 1990Gloss et al, 1987;Swift et al, 1987).…”