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

Abstract

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...

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

“…We recently demonstrated that 31E8^E2C is able to inhibit the growth of HeLa cells by binding to HPV promoter sequences and repressing the expression of the viral oncogenes E6 and E7 (38). To investigate whether the E8^E2C proteins of HPV16 and -18 also lead to growth arrest, HeLa cells were transfected with the pPur plasmid, providing puromycin resistance, and the eukaryotic expression vector pSG5 or derivatives expressing E8^E2C of HPV16, -18, or -31 in a 4:1 ratio.…”

“…To be able to monitor the expression levels of the different E8^E2C and E2 proteins, HA epitopes were inserted internally in HPV16 and HPV18 E2 and E8^E2C genes as previously described for 31E2 and 31E8^E2C (38). HeLa cells were transfected with the empty vector or the respective expression vectors encoding the different E8^E2C or E2 proteins.…”

“…The luciferase reporter plasmids pC18-SP1-luc, pGL 31URR-luc, pGL 31URR-BS2,3,4 mt-luc, and pGL 18URR-luc and the expression plasmids for HPV31 E8^E2C (pSG 31E8^E2C) hemagglutinin (HA)-tagged 31E8^E2C-HA, HPV31 E2, and HPV31 E2-HA have been previously described (38,39). To generate the luciferase reporter plasmid pGL 16URR-luc, a fragment (HPV16 nucleotides [nt] 7141 to 7173) was amplified by PCR using the cloned HPV16 114b genome (kindly provided by M. Dürst) as a template and primers 16URR XhoI-F (5Ј-ATATATCTCGAGTATTGTATGTATGT-3Ј) and 16URR NcoI-R (5Ј-ATATATCCATGGTGCAGTTCTCTTTTG-3Ј).…”

“…41665-062; Invitrogen, Karlsruhe, Germany) supplemented with gentamicin and 10% fetal bovine serum (Seromed Biochrom, Berlin, Germany). Colony reduction assays were essentially performed as previously described (38). Briefly, 3 ϫ 10 5 HeLa cells were transfected with Fugene HD (Roche) in 60-mm dishes with 0.8 g of the respective expression vectors and 0.2 g of pPur.…”

“…In contrast, the 31E8^E2C protein inhibits the growth of HeLa cells as well as 31E2 (31,38). Growth inhibition by 31E8^E2C was correlated with a reduction of E6/E7 transcripts and the induction of p53, p21, and pRb proteins (38).…”