Transcriptional Activity among High and Low Risk Human Papillomavirus E2 Proteins Correlates with E2 DNA Binding

Abstract

The full-length E2 protein, encoded by human papillomaviruses (HPVs), is a sequence-specific transcription factor found in all HPVs, including cancer-causing high risk HPV types 16 and 18 and wart-inducing low risk HPV types 6 and 11. To investigate whether E2 proteins encoded by high risk HPVs may function differentially from E2 proteins encoded by low risk HPVs and animal papillomaviruses, we conducted comparative DNAbinding and transcription studies using electrophoretic mobility shift assays and cell-free transcription systems reconstituted with purified general transcription factors, cofactor, RNA polymerase II, and with E2 proteins encoded by HPV-16, HPV-18, HPV-11, and bovine papillomavirus type 1 (BPV-1). We found that although different types of E2 proteins all exhibited transactivation and repression activities, depending on the sequence context of the E2-binding sites, HPV-16 E2 shows stronger transcription activity and greater DNA-binding affinity than those displayed by the other E2 proteins. Surprisingly, HPV-18 E2 behaves more similarly to BPV-1 E2 than HPV-16 E2 in its functional properties. Our studies thus categorize HPV-18 E2 and BPV-1 E2 in the same protein family, a finding consistent with the available E2 structural data that separate the closely related HPV-16 and HPV-18 E2 proteins but classify together the more divergent BPV-1 and HPV-18 E2 proteins.Human papillomaviruses (HPVs) 1 are a family of small DNA viruses that cause a wide variety of human diseases ranging from benign epithelial lesions, such as warts, to invasive cancers, such as cervical carcinoma. So far, more than 100 HPV types have been identified and fully sequenced, whereas more than 120 putative novel types have been partially characterized (1, 2). HPV types frequently found in invasive cancers include These are classified as high risk HPVs. In contrast, HPV types that are rarely found in cancers but are associated with genital warts, such as HPV-6 and HPV-11, are considered low risk HPVs (1-3). Because the genomic structures of HPVs are highly conserved, it is important to determine the functional differences among individual HPV gene products which lead to etiologically high and low risk phenotypes. Previous comparative studies have primarily focused on HPV-encoded E6-and E7-transforming proteins. These studies found that E6 and E7, from high risk HPVs, lead to cellular transformation much more readily than low risk E6 and E7 proteins (for review, see Refs. 4 and 5). In both high and low risk HPVs, expression of E6 and E7 is transcriptionally regulated via the E6 promoter by many cellular and viral proteins. The full-length viral E2 protein is a sequence-specific transcription factor that functions as an activator or repressor to regulate tightly the E6 promoter through four consensus E2-binding sites (E2-BSs), ACCGN 4 CGGT (6, 7), whose locations within the upstream regulatory region (URR) are highly conserved among genital HPVs. Efficient activation of the E6 promoter requires binding of E2 protein to the promoter...

E2 Proteins from High- and Low-Risk Human Papillomavirus Types Differ in Their Ability To Bind p53 and Induce Apoptotic Cell Death

“…1D). Previous studies have shown that, at equivalent expression levels, the LR-HPV E2 proteins activate transcription less well than the HR-HPV E2 proteins (21), and this is also seen with the tagged E2 proteins. The tagged proteins activate transcription to the same degree as the untagged proteins, and in the case of the HR-HPV16 E2 protein, the tagged LR-HPV E2 proteins block HPV16 E2-induced apoptosis.…”

TATA-binding Protein (TBP)-like Protein Is Required for p53-dependent Transcriptional Activation of Upstream Promoter of p21 Gene

“…1A). This difference may be due to limited amounts of functional intracellular p53 because p53 in HeLa cells is significantly repressed by papillomavirus proteins (45).…”

“…A gel shift assay was performed essentially in accordance with a method described by Hou et al (45). A DNA fragment spanning from Ϫ2266 to Ϫ2237 of the p21 gene that contains a p53-binidng sequence was used as a probe (see Fig.…”

Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

“…The removal of pol II activity from our purified Mediator complexes allowed us to unambiguously define the role of Mediator-P.5 and Mediator-P.85 on basal and activated transcription without further complications caused by minor pol II activity often found in Mediator preparations. For activator specificity, we also tested the effect of Mediator in supporting activation by the full-length human papillomavirus type 11 (HPV-11) E2 protein, which activates transcription from p2E2(IR)⌬53 template containing two copies of the E2-binding site located upstream of the adenovirus major late core promoter linked to a 280-nucleotide G-less cassette (36,94). As shown in Fig.…”