The Papillomavirus E8^∧E2C Protein Represses DNA Replication from Extrachromosomal Origins

Abstract

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8 ∧ E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8 ∧ E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8∧ E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8 ∧ E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8∧ E2C(1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8 ∧ E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.DNA viruses such as certain types of human papillomaviruses (HPV) and Epstein-Barr-virus (EBV), a member of the human herpesvirus family, are of major clinical interest because infections with these viruses have been linked to the development of human malignancies. HPV types 16, 18, 31, and 45 represent the predominant etiological agents of cervical cancer, and EBV infection has been suggested to contribute to the development of a number of lymphomas and nasopharyngeal carcinoma (28,42,43). Both HPV and EBV replicate as extrachromosomal elements in the nucleus of human cells. Interestingly, papillomaviruses and EBV maintain their genomes at defined copy numbers (10 to 50 virus copies per EBV-infected cell and 100 virus copies per papillomavirusinfected cell) in cultured cells over long periods of time (1,3,10,16,39,40). This behavior in tissue culture is thought to reflect viral DNA replication during persistent infections in vivo.In the latent phase, EBV DNA replication initiates at oriP, whereas in the lytic (productive) phase, oriLyt is responsible for the replication of EBV DNA (12,40). In contrast to oriLyt, the activity of oriP requires the viral EBNA1 protein, and viral copy number is kept constant by host cell replication licensing mechanisms, which ensure that eukaryotic genomes are copied exactly once before cell division (1, 40, 41). Most likely, this is achieved by recruiting the human origin recognition complex to oriP (5,8,31).Papillomaviruses use a different strategy to successfully maintain their DNA at defined copy numbers. In contrast to oriP, papillomavirus origins are not subject to replication licensing, but appear to use copy number control mechanisms, which are not fully understood (10). Two viral proteins are essential for the activation of papilloma...

The E8 ∧ E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes

“…7A, lanes 4, 5, 9, and 10, respectively). These data demonstrate that the initial amplifications of both HPV-16 and HPV-31 (as previously reported [61]) are similarly repressed by an E8 gene product. We then analyzed the capacities of the HPV-16 and HPV-31 wt and E8Ϫ mutant constructs to immortalize primary HFKs in three independent long-term transfections, using two distinct donor foreskins (Fig.…”

“…E8 ∧ E2 interferes with E2-dependent transcriptional activation by the full-length E2 proteins via competitive binding to conserved E2 binding sites in BPV-1 (T. Haugen, unpublished data) and HPV-31 (54). Similarly, HPV E8 ∧ E2 products can also inhibit plasmid replication (2,61). This study uses a newly developed complementation assay for HPV-16 replication to define the structure of the HPV-16 …”

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

“…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. This activity depends upon the E8 repression domain that is required for efficient binding of the E8 Ù E2C protein to the HPV18 promoter sequences in vivo as shown by chromatin immunoprecipitation assays.…”

“…27,32,56,58 Second, in contrast to E2, E8 Ù E2C represses transcription not only from promoter-proximal binding sites but also from promoter-distal binding sites. 39 Third, the E8 Ù E2C protein acts as a repressor of transcription and replication 18,39,40 whereas Brd4 has been reported to contribute also to the activation of transcription and replication by E2. 32,56,59 Therefore, it is likely that the E8 domain interacts with host cell proteins different from Brd4 that may enhance binding of E8 Ù E2C to chromatin and also contribute to transcriptional repression.…”

“…55 The E8 Ù E2C-KWK mutant protein retains the ability to inhibit E2 activity in transcription and replication assays and is able to reduce the activities of transfected HPV18 and 31 E6/E7 promoter constructs, but it was always less efficient than the wt E8 Ù E2C protein, which could be explained by a reduced binding to E2BS in vivo. 39,40 It has been described that E2 mutant proteins unable to inhibit the growth of HeLa cells show a reduced binding to the endogenous HPV18 promoter by in vivo footprint analyses. 31 A very recent publication describes that the interaction with the cellular Brd4 protein and the HPV11 E2 protein enhances binding of E2 to the endogenous HPV18 promoter DNA and chromatinized templates in vitro and that this interaction is required for promoter repression.…”