Human papillomavirus type 16 E6 protein inhibits DNA fragmentation via interaction with DNA fragmentation factor 40

The E6 oncoprotein of human papillomavirus (HPV) is critical in cervical cancer development. Using the yeast two-hybrid assay, we showed that HPV-16 E6 (16E6) interacts with one of the DNA fragmentation factors (DFFs), DFF40, which mediates DNA degradation during apoptosis. Furthermore, 16E6 interacts with DFF40 through its zinc finger motif 2 and a bridge section linking the two zinc finger motifs. DNA fragmentation assays disclosed that 16E6 binding to DFF40 leads to blockage of DNA cleavage. Our data collectively suggest that suppression of DNA fragmentation through 16E6–DFF40 interaction is a central event promoting tumorigenesis.     

HPV-16 E6 oncoprotein impairs the fidelity of DNA end-joining via p53-dependent and -independent pathways

We previously reported that the E6 oncoprotein of high-risk human papillomavirus (HPV) caused genetic instability and oncogenesis by disrupting cellular DNA repair. Here, to investigate the effect of different domains of E6 on DNA double-strand break (DSB) repair, we infected normal human oral fibroblasts (NHOF) with retroviruses expressing wild-type (wt) or mutant (mt) HPV-16 E6 and examined the cellular DNA end-joining (EJ) activity. The cells expressing E6 showed not only a diminution of error-free DNA EJ but also an increase in erroneous DNA EJ capacity if compared with cells without wt E6. Analysis of DNA EJ activities from the cells expressing mt HPV-16 E6 indicated that binding to p53 and the presence of both intact zinc finger domains of E6 are necessary for inducing the E6-mediated aberrant DNA EJ activity. Also, deletion of the PDZ binding C-terminal region reduced this activity by 50%. These findings suggest that E6 can disrupt the fidelity of DSB repair via both p53-dependent and -independent pathways and the impaired fidelity might contribute to the development of genetic instability found in HPV-associated cancer.     

The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes

Using a recently described keratinocyte assay, we demonstrate that HPV-18 DNA induces two progressive steps in cellular transformation (a large cell and a small cell stage). Both steps of this keratinocyte transformation can be achieved with a subgenomic fragment containing only the HPV-18 regulatory region and E6/E7 genes. Similar to cell lines transformed by the complete HPV-18 genome, keratinocytes transformed by the HPV-18 E6/E7 genes express the major early viral protein (E7) but are non-tumorigenic in nude mice. Interestingly, HPV-18 DNA was noted to be 5 times more efficient than HPV-16 DNA for in vitro keratinocyte transformation, regardless of the method of DNA transfection.     

ErbB4 (JM-b/CYT-1)-induced expression and phosphorylation of c-Jun is abrogated by human papillomavirus type 16 E5 protein

Human papillomavirus type 16 E5 (HPV-16 E5) is a highly hydrophobic membrane protein with weak-transforming activity, which is associated with ErbB4 receptor in HPV-16-infected cervical lesions. Presently, we investigated the transforming mechanisms of E5 involving ErbB4 signaling. Firstly, we report a role for ErbB4 (JM-b/CYT-1) receptor that activates c-jun gene expression and phosphorylating at Ser63 and Ser73 of the c-Jun protein in ligand-independent and Ras-c-jun NH(2)-terminal kinase-dependent pathway. Secondly, we show that HPV-16 E5 protein can form a complex with ErbB4 via binding to the extracellular and transmembrane domains of ErbB4 (JM-b/CYT-1). When co-expressing HPV-16 E5 and ErbB4 in cells, E5 can abrogate ErbB4-induced c-Jun protein expression and phosphorylation resulted in increasing cell proliferation compared to ErbB4-expressing cells. The interaction between of HPV-16 E5 and ErbB4 provides more insight into the mechanisms of HPV-16 E5 transformation induction.     

E1A deregulates the centrosome cycle in a Ran GTPase-dependent manner

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH(2)-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH(2)-terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A. These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability.     

HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation

It has recently been shown that the high-risk human papillomavirus (HPV) E6 proteins can target the PDZ-domain containing proteins, Dlg, MUPP-1, MAGI-1 and hScrib for proteasome-mediated degradation. However, the E6 proteins from HPV-16 and HPV-18 (the two most common high-risk virus types) differ in their ability to target these proteins in a manner that correlates with their malignant potential. To investigate the underlying mechanisms for this, we have mutated HPV-16 and HPV-18 E6s to give each protein the other's PDZ-binding motif. Analysis of these mutants shows that the greater ability of HPV-18 E6 to bind to these proteins and to target them for degradation is indeed due to a single amino acid difference. Using a number of assays, we show that the E6 proteins interact specifically with only one of the five PDZ domains of MAGI-1, and this is the first interaction described for this particular PDZ domain. We also show that the guanylate kinase homology domain and the regions of MAGI-1 downstream of amino acid 733 are not required for the degradation of MAGI-1. Finally, in a series of comparative analyses, we show that the degradation of MAGI-1 occurs through a different mechanism from that used by the E6 protein to induce the degradation of Dlg and p53.     

The RING domain of Mdm2 can inhibit cell proliferation

Mdm2 is a p53-inducible phosphoprotein that negatively regulates p53 by binding to it and promoting its ubiquitin-mediated degradation. Alternatively spliced variants of Mdm2 have been isolated from human and mouse tumors, but their roles in tumorigenesis, if any, remain elusive. We cloned six alternatively spliced variants of Mdm2 from E(mu)-Myc-induced mouse lymphomas, all of which lacked the NH(2)-terminal p53-binding domain but conserved the remainder of the Mdm2 protein. Enforced expression of full-length Mdm2 in primary mouse embryo fibroblasts or bone marrow-derived, interleukin 7-dependent pre-B cells accelerated their proliferation, whereas unexpectedly, overexpression of truncated Mdm2 isoforms inhibited their growth. Truncated variants were active as inhibitors whether they localized predominantly to the nucleus or cytoplasm. Despite the absence of the p53-binding domain, growth inhibition remained strictly p53 dependent (but not p19(Arf) dependent) and could be overcome by full-length Mdm2. The intact RING finger domain at the Mdm2 COOH terminus (amino acids 399-489) was necessary and sufficient for growth inhibition by truncated Mdm2 proteins and could physically interact with either the RING finger domain or central acidic region of full-length Mdm2. However, such interactions do not inhibit Mdm2 E3 ubiquitin ligase activity in vitro using p53 as a substrate. Expression of growth-inhibitory Mdm2 isoforms in tumors remains an enigma.     

E6 protein of human papillomavirus type 18 binds zinc

The E6 open reading frames of human and animal papillomaviruses encode a transforming protein containing a conserved pattern of repeating cysteine doublets (cys-x-x-cys) similar to that found in steroid receptor zinc finger proteins. The spacing between the cysteine doublets, however, is twice as long as in any other zinc finger protein. To demonstrate that an E6 protein could indeed bind zinc, we synthesized the human papillomavirus type 18 E6 protein in insect cells with a baculovirus vector and analysed the protein for zinc-binding activity by a zinc-blot assay. Probing of E6 protein blotted to nitrocellulose from SDS-polyacrylamide gels with radioactive [65Zn]Cl2 demonstrated that it possessed zinc-binding activity. Reduction of cysteines with DTT prior to the addition of zinc greatly increased the zinc-binding activity of blotted E6 protein. Competition experiments showed that Cd2+ and Co2+ were more effective competitors for zinc-binding than Mg2+ or Ca2+, indicating that zinc atoms may be tetrahedrally coordinated in E6-zinc complexes. We mapped zinc-binding protein domains by proteolytic cleavage and demonstrated that a small 4kDa fragment of the protein retained zinc-binding activity, consistent with a model of individual 29 amino acid zinc-binding 'fingers'.     

The NH(2)-terminal and conserved region 2 domains of adenovirus E1A mediate two distinct mechanisms of tumor suppression.

Adenovirus E1A has been shown to suppress tumor growth and induce apoptosis in response to stress. To determine the mechanisms and regions of E1A that mediate these functions, we characterized stable transfectants of various E1A mutants in murine melanoma cells both in vitro and in vivo. Three E1A-mutant constructs were used in this study, those having a single deletion at either the NH(2)-terminal (dl1101) or conserved region 2 (CR2) domain (dl1108), or double deletions at both domains (dl0108). The in vitro study showed that the CR2 domain is required for E1A-mediated apoptosis, whereas the NH(2)-terminal domain is dispensable. The in vivo study showed that dl1101 and dl1108 were still able to suppress tumor growth, whereas dl0108 lost tumor-suppressive activity. By in situ immunohistostaining, we found that factor VIII, a marker for angiogenesis, was greatly suppressed in dl1108 transfectants that are resistant to apoptosis. Thus, inhibition of angiogenesis is involved in the NH(2)-terminal domain of E1A. In conclusion, we suggest that the NH(2)-terminal and CR2 domain of E1A mediate two distinct mechanisms of tumor suppression.     

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.