Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8ˆE2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1␤/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1␤/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1␤/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8ˆE2C.Persistent infections with certain human papillomavirus (HPV) types are a necessary risk factor for the development of cervical cancer (6). Papillomavirus genomes replicate as nuclear plasmids with ϳ100 copies per cell in undifferentiated keratinocytes, and viral copy number increases substantially upon induction of differentiation of the host cell (38). Viral proteins derived from the E1 and E2 genes function as sequence-specific DNA binding proteins and are involved in the initiation of DNA replication, control of viral transcription, and segregation of viral genomes (20,21,27,35). The E1 protein represents the viral replication initiator protein and acts as a replicative hexameric helicase (35). The viral E2 protein is a sequence-specific DNA binding protein that recruits the E1 protein to the viral replication origin by proteinprotein and protein-DNA interactions (35). In addition, E2 is a transcriptional modulator with opposing activities: E2 represses the viral E6 promoter from promoter-proximal E2 binding sites (E2BS) but can also strongly activate synthetic promoters from distal sites (21). Transactivation activity is mediated by the interaction of the E2 amino terminal domain with cellular proteins such as AMF1/Gps1, p300/CBP, Brd4, and cNAP1 (2,12,17,23,28,32).In addition to E2, several papillomaviruses express a second protein derived from the E2 gene, named E8ˆE2C, in which the E8 gene replaces the E2 activation domain that is responsible for transcriptional control and the activation of DNA replication (5,8,13,26,34,37). The E2C domain common to E2 and E8ˆE2C mediates dimeriz...

Expression of the E6 and E7 oncogenes of high-risk human papillomaviruses (HPV) is controlled by cellular transcription factors and by viral E2 and E8 ∧ E2C proteins, which are both derived from the HPV E2 gene. Both proteins bind to and repress the HPV E6/E7 promoter. Promoter inhibition has been suggested to be due to binding site competition with cellular transcription factors and to interactions of different cellular transcription modulators with the different amino termini of E2 and E8 ∧ E2C. We have now identified the cellular chromodomain helicase DNA binding domain 6 protein (CHD6) as a novel interactor with HPV31 E8 ∧ E2C by using yeast two-hybrid screening. Pull-down and coimmunoprecipitation assays indicate that CHD6 interacts with the HPV31 E8 ∧ E2C protein via the E2C domain. This interaction is conserved, as it occurs also with the E8 ∧ E2C proteins expressed by HPV16 and -18 and with the HPV31 E2 protein. Both RNA knockdown experiments and mutational analyses of the E2C domain suggest that binding of CHD6 to E8 ∧ E2C contributes to the transcriptional repression of the HPV E6/E7 oncogene promoter. We provide evidence that CHD6 is also involved in transcriptional repression but not activation by E2. Taken together our results indicate that the E2C domain not only mediates specific DNA binding but also has an additional role in transcriptional repression by recruitment of the CHD6 protein. This suggests that repression of the E6/E7 promoter by E2 and E8 ∧ E2C involves multiple interactions with host cell proteins through different protein domains.Persistent infections with "high-risk" human papillomaviruses (HPV) are a major risk factor for cervical cancer development (5, 10, 86). Carcinogenic HPV encode the E6 and E7 oncoproteins, which are required for the immortalization of normal human keratinocytes and the continous growth of cervical cancer cell lines, such as HeLa (40, 41). Expression of E6 and E7 transcripts is controlled by both cellular and viral transcription factors (70). Papillomavirus proteins derived from the E2 gene are involved in control of viral transcription, of DNA replication, and of segregation of viral genomes (29,37,38). The E2 open reading frame gives rise to multiple gene products by alternative RNA splicing. In addition to the full-length E2 protein, cells infected with bovine papillomavirus type 1 (BPV1), cottontail rabbit papillomavirus (CRPV), and HPV types 1, 11, 16, 31, and 33 express an E8 ∧ E2C transcript in which the small E8 domain is fused to the C-terminal domain of E2 (E2C) (9,18,28,46,52,58,63). Inactivation of E2 in the HPV16 genome increases E6/E7 transcription (59). Mutation of E8 ∧ E2C in the HPV31 or HPV16 genome increases genome copy number and E6/E7 transcripts, strongly suggesting that transcriptional repression by E8 ∧ E2C plays an important role in viral replication (31,63,85).The C-terminal domain (E2C) which is shared by E2 and E8 ∧ E2C is responsible for sequence-specific recognition of ACCN 6 GGT DNA sites (E2 binding site [E2BS]) and homoand he...