The E8 Domain Confers a Novel Long-Distance Transcriptional Repression Activity on the E8^E2C Protein of High-Risk Human Papillomavirus Type 31

Abstract

Infections with high-risk human papillomaviruses (HPVs) are the major risk factor for the development of anogenital cancers. Viral E2 proteins are involved in viral DNA replication and regulation of transcription. Repression of the viral P97 promoter by E2 proteins has been implicated in the modulation of the immortalization capacity and DNA replication properties of high-risk HPVs. Analysis of the cis and trans requirements for repression of the HPV type 31 (HPV31) P97 promoter, however, revealed striking differences between the full-length E2 and the E8ˆE2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8ˆE2C completely inhibited the P97 promoter from a single promoter-distal E2 binding site. This novel long-distance repression activity of the E8 domain also enabled E8ˆE2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8ˆE2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8ˆE2C might contribute to the progression of high-risk HPV-induced lesions.The replication cycle of human papillomaviruses (HPVs) can be divided into two stages. In the basal cell layer of the epidermis, which is composed of division-competent keratinocytes, HPVs establish a persistent, nonproductive infection in which viral DNA genomes are replicated as extrachromosomal elements at low levels and only early viral genes are transcribed. The productive viral replication cycle occurs upon differentiation of infected keratinocytes, which results in highlevel replication of viral genomes, induction of late-gene transcription, and synthesis of infectious virions (22,49).Infections with a subset of HPV types dramatically increase the risk for the development of malignancies of the anogenital tract, and these types have been designated as high-risk HPVs (56,57). Within this group, high-risk HPV type 16 (HPV16), HPV18, and HPV31 have been most intensively studied at the molecular level. Expression of the early E6 and E7 gene products of high-risk HPVs is sufficient to immortalize normal human keratinocytes (NHKs), the natural target cells for HPVs (34). This property is not shared by E6 and E7 genes from low-risk HPV6 and HPV11 and is therefore regarded as being relevant to the carcinogenic potential of high-risk HPVs (34). The exact molecular events that lead to malignant progression of high-risk HPV-induced lesions are still largely unknown.Several lines of evidence suggest that transcriptional modulation of early viral gene expression is a central regulatory event. In the absence of viral gene products, HPV early-gene transcription is activated by a variety of host cell transcription factors, which interact with regulatory sequences located upstream of the major early promoter of high-risk HPV16, -18, and -31, designated P97 for HPV16 and -31 and P105 for HPV18 (22). The basal activity of HPV early promoters can be further modulated by viral E2 proteins, which are s...

The E8 repression domain can replace the E2 transactivation domain for growth inhibition of HeLa cells by papillomavirus E2 proteins

“…Luciferase assays were carried out 48 hr after transfection as previously described. 39 The data presented in the figures are the average of at least 3 independent transfection experiments carried out in duplicate.…”

“…39,41 An HA-tagged version of E2 was constructed by inserting a double stranded oligonucleotide (5 0 -AATTCTACC CATACGATGTTCCAGATTACGCTGAG-3 0 ) into the EcoRI site of the HPV31 E2 sequence in the context of pSXE2. Sequences encoding the HA epitoptag were inserted in the E8 Ù E2C and mutant genes into the EcoRI site by PCR.…”

“…29,42 The luciferase reporter plasmid pC18-SP1-luc has been described. 39 To generate the luciferase reporter plasmid p18URR-luc, a fragment (HPV18 nt. 7127-105) was amplified by PCR using the cloned HPV18 genome as template and primers 18URR MluI for (5 0 -TGTGCGTGTACGCGTCAGGAAGTAAT-3 0 ) and 18URR NcoI rev (5 0 -TCAAAGCGCGCCATGGTATT GTGG-3 0 ).…”

“…18 In contrast to E2, E8 Ù E2C is also able to repress transcription from promoter-distal E2BS and this is dependent upon the E8 part of the protein. 39 The E8 domain …”

“…18 In contrast to E2, E8 Ù E2C is also able to repress transcription from promoter-distal E2BS and this is dependent upon the E8 part of the protein. 39 The E8 domain represents a transferable transcription modulation domain and can confer repression activity to the Gal4 DNA binding domain. 40 In this report, we demonstrate that E8 Ù E2C protein derived from HPV31 is able to inhibit the growth of HeLa cells by repressing the endogenous HPV18 E6/E7 promoter and reactivating dormant tumor suppressor pathways.…”