Chelating agents stabilize the monomeric state of the zinc binding human papillomavirus 16 E6 oncoprotein

The E6 protein of human papillomavirus 16 is known to be difficult and, when overexpressed, insoluble and agglomerated. It has two putative zinc ion binding sites crucial for its function. No metallochaperone has yet been found to deliver zinc ions to the E6 protein. Here, we report that a specific chelating agent, which we think functionally mimics a metallochaperone, stabilized the soluble monomeric form of E6 and inhibited multimerization in vitro. This effect seemed to depend on the chelating strength of the agent. While strong chelating agents precipitated the E6 protein and weak chelating agents did not favor the monomeric form of E6, chelating agents of intermediate strength [L-penicillamine and ethylene glycol bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA)] effectively support the formation of a monomer. We did not observe formation of a dimer or defined oligomers. Degradation assays imply that the monomer is the biologically active form of the protein. Since EGTA favors the formation of monomeric over agglomerated E6 protein, we propose that chelating agents of appropriate strength could assist zinc delivery to recombinant metalloproteins in vitro and may even destabilize existing agglomerates.     

Golgi alkalinization by the papillomavirus E5 oncoprotein

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.     

PKN binds and phosphorylates human papillomavirus E6 oncoprotein

The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two viral oncoproteins E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary epithelial cells has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.     

Bovine papillomavirus E7 oncoprotein inhibits anoikis

The bovine papillomavirus type 1 (BPV-1) E7 oncoprotein is required for the full transformation activity of the virus. Although BPV-1 E7 by itself is not sufficient to induce cellular transformation, it enhances the abilities of the other BPV-1 oncogenes to induce anchorage independence. We have been exploring the mechanisms by which E7 might affect the transformation efficiency of other viral oncoproteins and in particular whether it might protect cells from apoptosis. We report here that BPV-1 E6 and E7 can each independently inhibit anoikis, a type of apoptosis that is induced upon cell detachment. Using site-directed mutagenesis, we determined regions of the E7 protein that were essential for its antiapoptotic activity. The ability of E7 to inhibit anoikis did partially correlate with an ability to enhance anchorage independence of BPV-1 E6-transformed cells. In addition, the antiapoptotic activity of E7 also only partially correlated with its ability to bind p600, a cellular protein that has previously been reported to play a role in anoikis. We conclude that the contribution of E7 to BPV-induced cellular transformation may involve its ability to inhibit anoikis but that additional functional activities must also be involved.     

Human papillomavirus 16 E6 oncoprotein interferences with insulin signaling pathway by binding to tuberin

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either TSC1 or TSC2 tumor suppressor gene. TSC1 and TSC2 products, Harmatin and Tuberin, form the functional complex to serve as the negative regulator for insulin-induced phosphorylation of S6 kinase and elF4E-binding protein 1. High-risk human papillomavirus (HPV) infection is the necessary cause for cervical cancer. E6 oncoprotein encoded by HPV plays a pivotal role in carcinogenesis by interference with the host intracellular protein functions. In this study, we show that HPV16 E6 interacts with tumor suppressor gene TSC2 product, Tuberin, and results in the phosphorylation of S6 kinase and S6 even in the absence of insulin. The overexpression of Tuberin overcomes the effect of E6 on S6 kinase phosphorylation. Binding with HPV16 E6 causes the proteasome-mediated degradation of Tuberin. A DILG motif and an ELVG motif located in the carboxyl-terminal of Tuberin are required for E6 binding. In addition, the Tuberin interaction region in E6 has been mapped in the amino-terminal portion of HPV16 E6, which is different from the binding domain with p53. These results provide a possible link between E6-induced oncogenesis and the insulin-stimulated cell proliferation signaling pathway.     

Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.     

The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53

The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.     

Apoptosis of mortal human fibroblasts transformed by the bovine papillomavirus E5 oncoprotein

Mortal human fibroblasts can be partially transformed by the bovine papillomavirus E5 oncoprotein through activation of the platelet-derived growth factor beta receptor. Here, we report that these cells undergo massive apoptosis 2 weeks after confluence. Although activation of caspase 3 was observed in the apoptotic cells, it was not required for apoptosis. The appearance of the mitochondrial proteins cytochrome c and apoptosis-inducing factor in cytosolic and nuclear compartments, respectively, provided a basis for mitochondrial dysfunction and a caspase-independent mechanism of apoptosis in these cells. Although an activating conformational change in Bax also was evident in the apoptotic cells, enforced overexpression of Bcl-2 was insufficient to prevent apoptosis. Finally, a small peptide present in the conditioned medium from dying transformed cells appeared responsible for inducing apoptosis through affecting a conformational change in Bax and eventual relocalization of apoptosis-inducing factor to the nucleus. Thus, an atypical apoptotic pathway is activated in mortal human fibroblasts in response to transformation induced by sustained receptor tyrosine kinase activation.     

Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein.

The 44-amino acid bovine papillomavirus E5 protein induces tumorigenic transformation of immortal rodent fibroblasts by binding to and activating the platelet-derived growth factor beta receptor (PDGFbetaR). Here E5 was expressed in mortal human diploid fibroblasts (HDFs), which lack the accumulated genetic changes that are present in immortal rodent cells. E5 induced focus formation and morphological transformation of HDFs without inducing anchorage independence or immortalization. Similar effects were observed with the v-sis and neu* oncogenes. E5-PDGFbetaR complexes were observed in the E5-expressing HDFs, as was constitutive PDGFbetaR activation, which was required for the transforming activity of E5. The E5 HDFs attained a higher saturation density than the control cells, expressing increased levels of hyperphosphorylated retinoblastoma protein at subconfluent densities. However, when these cells reached confluence, growth inhibition accompanied by dramatic down-regulation of the PDGFbetaR, and retinoblastoma protein was induced apparently by a factor secreted into the medium. This may represent a novel negative feedback mechanism controlling PDGFbetaR-induced proliferation and thereby protecting against complete transformation.     

Chaperone holdase activity of human papillomavirus E7 oncoprotein

E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.