Inhibition of Transcription and DNA Replication by the Papillomavirus E8⁁E2C Protein Is Mediated by Interaction with Corepressor Molecules

Abstract

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...

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

“…While 31E8^E2C was also found to interact with TRIM28 and SETDB1 (1), siRNA knockdown experiments indicated that these proteins do not contribute to the repression of the HPV URR by 31E8^E2C (31). Furthermore, while HDAC inhibitors relieved repression by 31E8^E2C, this was not the case for the KOX1 KRAB domain (1,24). These data suggest that the repression of the URR by E8^E2C and KRAB-E2C is achieved by the recruitment of different transcription factors and possibly by different repression pathways.…”

“…The transcriptional-repression activity of E2 has been linked to the interaction with the cellular Brd4, SMCX, and EP400 proteins (35,45). In contrast, the transcriptionalrepression activity of 31E8^E2C has been linked to the interaction of the E8 domain with cellular corepressors, such as HDAC3 and the N-CoR protein (1,31). The KRAB domain of KOX1 represses transcription through the recruitment of the corepressor TRIM28/KAP1/TIF1␤/KRIP1, heterochromatin protein 1 (HP1) alpha, HP1 gamma, and the histone methyltransferase SETDB1 (12,23,25,34).…”

“…Genetic and biochemical analyses of the 31E8^E2C protein have demonstrated that the 31E8 domain is required for transcriptional repression. This is due to the recruitment of cellular corepressors, such as the histone deacetylase 3 (HDAC3)/N-CoR complex, by the 31E8 domain (1,31). The analysis of E8^E2C functions in the context of HPV16, the most potent cancer-inducing HPV, has revealed differences from HPV31.…”

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.…”

The Role of E2 Proteins in Papillomavirus DNA Replication

“…The shorter E2s antagonize the function of full-length E2 by competing for E2 DNA binding sites. In addition, the E8 of HPV 31 E8/E2 protein itself is a transcriptional repressor domain that functions independently of binding site competition inhibiting transcription and DNA replication by interacting with corepressor molecules such as NCoR1/HDAC3, the histone methyltransferase SETDB1, and the TRIM28 protein (Ammermann et al, 2008;Powell et al, 2010). The full-length and truncated E2 proteins are able to form dimers through their common Cterminal DNA-binding-dimerization domain (McBride et al, 1989).…”