Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8؊), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8؊ and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. P ersistent infections with high-risk (HR) human papillomaviruses (HPV) are a necessary risk factor for the development of cancer of the cervix uteri and are also responsible for a fraction of anal, vaginal, penile, and oropharyngeal cancers (1-3). Notably, HPV16 is an extremely powerful human carcinogen that is responsible for approximately 50% of cervical cancers and is also represented in other HR-HPV-related cancers at high frequencies (2).HPV have a double-stranded DNA genome that is covalently closed and organized in nucleosomes. HPV possess eight to nine open reading frames (E1, E2, E4 to E8, L1, and L2) that are transcribed into polycistronic messages which are further processed by alternative splicing (4). It is currently assumed that HR-HPV encode at least 10 different viral proteins.The HR-HPV replication cycle is tightly linked to the differentiation state of the infected epithelium. In undifferentiated basalcell-like cells, HR-HPV genomes are maintained in the nucleus at 10 to 100 extrachromosomal copies per cell, and only early genes, mainly transcribed from the major early viral promoter located immediately upstream of the E6 gene (P97 for HPV16), but not the late viral L1 and L2 genes are expressed (5). Replication of the viral genome in undifferentiated cells requires the viral E1 and E2 proteins (6). E1 and E2 are sequence-specific DNA binding proteins which form a complex that recognizes the viral origin of replication (6). E1 assembles into a hexamer that acts as the replicative helicase and unwinds the viral DNA...

Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

Continuous expression of the human papillomavirus (HPV) oncoproteins E6 and E7 is required for the growth of cervical cancer cell lines. So far, only the overexpression of the wild type papillomavirus E2 protein has been shown to induce growth arrest in HPV18-positive HeLa cells by repressing E6/E7 transcription. Growth arrest by E2 requires the aminoterminal transcription activation domain in addition to the carboxyterminal DNA-binding domain. Several papillomaviruses such as the carcinogenic HPV31 express in addition to E2 an E8 Ù E2C fusion protein in which the E8 domain, which is required for repression of replication and transcription, replaces the E2 activation domain. Infections with certain human papillomaviruses (HPV), most notably types 16 and 18, are a necessary risk factor for the development of cervical cancer.1 While the HPV genome is present in an episomal state in low-grade lesions, the whole viral genome or fragments thereof are often integrated into the chromosomal DNA of the host cell in the majority of HPV-induced carcinomas.2 The HeLa cell line was derived from a cervical cancer and contains a partial HPV18 genome integrated into the host chromosomes. 4-7 The E6 and E7 proteins derived from carcinogenic HPVs interfere with cell cycle control proteins. E6 forms a ternary complex with the E3 ubiquitin ligase E6AP and p53, which results in the degradation of p53 and therefore in the reduction of mRNA levels of p53 target genes. 8,9 The E7 protein binds to Rb family members and disrupts Rb/E2F complexes, resulting in increased expression of E2F-regulated genes. 10,11 In addition, the E7 protein enhances degradation of Rb family members. 12,13 A peculiar feature of carcinogenic HPV types is that the E6 and E7 transcripts are generated by alternative splicing from a common precursor RNA.14 Transfection studies using reporter plasmids of the corresponding promoters of HPV16, 18 or 31 demonstrated that coexpression of viral E2 proteins resulted in repression of promoter activity, which is due to binding of E2 to proximal recognition sequences. [15][16][17][18][19] Similarly, repression of the endogenous HPV18 E6/E7 promoter in HeLa cells could be observed when E2 genes from bovine papillomavirus Type 1 (BPV1), HPV16 or 18 were introduced into HeLa cells. [20][21][22] Overexpression of E2 resulted in growth arrest or apoptosis of HPV-positive cervical cancer cells. [20][21][22] This was mainly due to the specific repression of the HPV promoter as the growth of HPV negative cells was not influenced by overexpression of E2. [20][21][22][23][24] In line with this, the reduction of E6/E7 mRNA levels resulted in an increase in E6 and E7 target protein levels such as p53, p21 and pRb. [20][21][22][23][24] E2 proteins bind as dimers to specific DNA sequences (E2 binding site, E2BS) to modulate transcription and viral DNA replication. The carboxyterminal domain which comprises 100 amino acids (aa) is sufficient for sequence-specific DNA recognition and dimerization 25 (Fig. 1). The E2 aminoterminal domain of...

Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells. IMPORTANCEHPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

Infections with certain human papillomaviruses (HPV), such as type 16 (HPV16), 18, or 31, are a necessary risk factor for the development of cervical cancer. Transcript analyses of several HPV revealed that the viral E2 gene encodes both the E2 regulator protein and the E8^E2C protein, which differ in their amino termini. Up to now, functional studies have focused on HPV31 E8^E2C and demonstrated that it is a potent repressor of viral transcription and replication. However, recent analyses of HPV16 genomes have suggested that E8^E2C proteins may differ in their activities. Therefore, we performed a comparative analysis of E8^E2C proteins of HPV16, -18, and -31. All E8^E2C proteins potently inhibited HPV E6/E7 oncogene promoters, and also displayed long-distance transcriptional-repression activities. Furthermore, the expression of all E8^E2C proteins inhibited the growth of HeLa cells. Expression of E8^E2C proteins rapidly increased the protein levels of the E6 and E7 targets p53 and p21, consistent with the repression of the endogenous HPV18 E6/E7 promoter. All E8^E2C proteins induced G 1 arrest more efficiently than E2 proteins and activated senescence markers. Furthermore, we demonstrate that the 31E8 domain can be functionally replaced by the KRAB repression domain derived from KOX1. The KRAB-E2C fusion protein possesses long-distance transcriptional-repression activity and inhibits the growth of HeLa cells comparably to E8^E2C. Taken together, our results suggest that the E8^E2C proteins of HPV16, -18, and -31 are highly conserved transcriptional repressors that inhibit the growth of HeLa cells by repression of E6/E7 transcription but do not have proapoptotic activities.Persistent infections with human papillomaviruses (HPV), such as HPV type 16 (HPV16), -18, or -31, are a necessary risk factor for the development of invasive cervical cancer (4, 42, 47). HPV16 accounts for ϳ55%, HPV18 for ϳ16%, and HPV31 for only ϳ4% of cervical cancers worldwide (3), but the underlying differences accounting for these behaviors are not well understood. The viral E2 gene expresses crucial regulatory proteins involved in replication, transcription, and maintenance of viral genomes (19,40). The E2 protein is a sequence-specific DNA binding protein that recognizes four E2 binding sites (E2BS) upstream of the HPV E6/E7 promoter through its C-terminal domain (E2C) (26). The amino-terminal domain of E2 (E2TA) is responsible for the activation of transcription, the activation of viral replication, and attachment to mitotic chromosomes (19,40). In addition to E2, several HPV express a spliced RNA that expresses a fusion protein consisting of the small E8 domain fused to E2C (9,29,33,36,37). The functions of the E8^E2C protein have been mainly investigated with HPV31. It was shown that E8^E2C knockout HPV31 genomes displayed a strong overreplication of viral genomes in short-term analyses (37). Despite this, in stable cell lines, HPV31 E8^E2C (31E8^E2C) knockout genomes were not maintained as episomes but only found integrated into t...