Interaction of the Papillomavirus E8∧E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression

Abstract

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...

Growth Inhibition of HeLa Cells Is a Conserved Feature of High-Risk Human Papillomavirus E8^E2C Proteins and Can Also Be Achieved by an Artificial Repressor Protein

“…S1 in the supplemental material), which has been demonstrated by single-amino-acid mutations (W6A and K7A) to be sufficient for repressing the replication of HPV31 genomes (39,46). This also suggests that the repression activities of all ␣-HPV E8^E2C proteins might be due to a conserved interaction of the E8 domain with an HDAC3/NCoR complex, as demonstrated for 31E8^E2C, and also to interaction with the CHD6 protein via the E2C domain (11,31).…”

“…Recent studies have indicated that transcriptional repression of the HPV URR by the E2 and 31E8^E2C proteins is due to interaction with different sets of cellular proteins via the amino termini and to a conserved interaction of the E2C domain with CHD6 (11). The transcriptional-repression activity of E2 has been linked to the interaction with the cellular Brd4, SMCX, and EP400 proteins (35,45).…”

Characterization of the nuclear matrix targeting sequence (NMTS) of the BPV1 E8/E2 protein — the shortest known NMTS

“…The HPV31 E8/E2 protein represses transcription and extra-chromosomal replication; many interactions responsible for these repressive functions are described. [41][42][43] However, the HPV31 E8 and BPV1 E8 proteins belong to different E8 clades (Puustusmaa and Abroi, manuscript in preparation). In addition, the subcellular localization of HPV31 E8/E2 has not yet been studied.…”

Human Papillomavirus

“…Ayrıca, HPV-1 ve HPV-31'de viral transkripsiyon ve replikasyonun bir negatif düzenleyici olarak E8-E2 füzyon proteini belirlenmiştir 8,9 . E8 geninin bir parçası olan E8-E2 füzyon proteini, E2 geninin C terminaline bağlanmış olup, viral transkripsiyonun alternatif bir yolunu oluştur-maktadır 16 . E8-E2C'nin viral yaşam siklusunun erken evresi boyunca HPV DNA replikasyonunun negatif bir düzenleyicisi olduğunu düşünülmektedir 17,18 .…”