Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53

E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins. BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL. E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation. Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP. In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex. 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53. Such 16E6 mutations were typically within the amino-terminus of 16E6. Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6. However, the interaction of 16E6 with E6AP was complex. While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation. In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation. These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way. These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.     

Properties of bovine papillomavirus E1 mutants.

Ostensibly comparable mutants of bovine papillomavirus type 1 (BPV-1) affecting the E1 open reading frame that were constructed in several laboratories have been reported to exhibit either reduced or increased transformation efficiencies in established mouse cell lines relative to wild-type BPV-1 DNA. To resolve these discrepancies, we have reexamined many of the mutants in mouse C127 cells by using focus formation assays. Our primary conclusions are that all E1 mutants tested consistently generated reduced numbers of transformants and that the reduced transformation was not due to cell toxicity associated with E1 mutations, as had been proposed. Our results can best be explained by the inability of the E1 mutants to replicate extrachromosomally, therefore leading to a rapid loss of the BPV-1 DNA and consequently, reduced transformation. In support of this hypothesis, we demonstrated that the human papillomavirus type 11 E1 protein was able to suppress BPV-1 transformation, probably because of interference with BPV-1 replication. Therefore, we attribute the phenotypic disparities reported by the various laboratories to still undefined differences in assay conditions.     

Changing the spacing between metal-binding motifs decreases stability and transforming activity of the human papillomavirus type 18 E7 oncoprotein

Spacing composed of 29 amino acids (AAs) between a pair of metal-binding motifs, Cys-X-X-Cys, is common to human papillomavirus (HPV) zinc-binding oncoproteins E6 and E7 from various HPV types. From the HPV 18 E7 gene encoding a 105-AA protein with the motifs at AAs 65-68 and 98-101, we constructed expression plasmids for two mutants, 18del73 and 18ins84, with varied spacing between the motifs. Mutant proteins 18del73 and 18ins84 had a deletion from AAs 74 to 88 and an insertion of three AAs substituting for AA 85, respectively. The mutations lowered the efficiency of E7-mediated focal transformation of rat 3Y1 cells, approximately parallel to the reduced level of steady-state E7 expressed in COS-1 cells. It is likely that, besides the presence of the motifs, the conserved spacing between the motifs is important for a stable structure of E7, but is not essential for the E7-transforming activity.     

The DNA-binding domain of HPV-16 E2 protein interaction with the viral enhancer: protein-induced DNA bending and role of the nonconserved core sequence in binding site affinity

We expressed the carboxy-terminal portion of the E2 open reading frame (ORF)-encoded protein of human papillomavirus type 16 (HPV-16) and purified it to near homogeneity. Using DNase I footprinting techniques, we show that like the homologous protein from bovine papillomavirus type 1 (BPV-1), HPV-18, HPV-11, it binds DNA at the enhancer consensus motif ACCN6GGT. Base and phosphate backbone contact points were determined using methylation protection and interference and ethylation interference assays. This HPV-16 E2 DNA-binding domain protein contacts the site at the outermost conserved GG residues which is similar to the interaction of the BPV-1 E2 system. However, there are many fewer phosphate backbone contacts. Using gel retardation assays, the HPV-16 E2 protein interaction with the consensus motif was characterized further based on the specific sequence of the noncontacted, nonconserved internal bases. Affinity of this E2 protein for the consensus site increased dramatically with an A.T-rich core sequence. Like the homologous BPV-1 protein, HPV-16 E2 protein induces DNA bending at its binding site. Furthermore, examination of the DNA region containing a single consensus motif far upstream from the major promoter, P97, revealed naturally bent DNA that was further bent upon interaction with the HPV-16 E2 protein.     

Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif.

The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136, and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. Immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-66 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 cells was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-66 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 16 E7 transformation of rat 3Y1 cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6.     

Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle

The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another alpha-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span.     

Isolation of cellular revertants from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16.

Three revertants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revertants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.     

Identification of H-2Kb Binding and Immunogenic Peptides from Human Papilloma Virus Tumour Antigens E6 and E7

Peptides can be used to induce MHC class I restricted cytotoxic T cells (CTL) tbrough in vivo immunization. This approach may enable the development of peptide vaccination schemes for immunization against viral infection in humans. Human papillomavirus (HPV) is one of a few viruses associated with human cancer and the development of an anti-cancer vaccine seems possible. As a model approach, we searched the E6 and E7 proteins of the human papillomavirus type 16 for possible murine MHC class I restricted peptide epitopes. We utilized the mouse H2-K^b peptide binding motif which consists of phenylalanine or tyrosine at position five and leucine at the carboxy-terminus with the modification that leucine could be replaced by other aliphatic but non-aromatic amino acids. Four peptide sequences from E6 and two from E7 were selected. These peptides were tested for their ability to bind and stabilize K^b and for their immunogenicity in vivo . It was shown tbat one peptide from E6, E6.1 (50–57), bound K^b, but was not able to prime mice in vivo . In contrast, the two selected E7 peptides E7.1 (21–28) and E7.2 (48–55) bound K^b and were immunogenic in vivo . The peptide induced CTL lysed syngeneic EL-4 cells transfected with the open reading frame of E7 but not vector only transfectants. This implies tbat both peptides were naturally processed and presented by K^b on the surface of target cells. MHC class I peptide binding motifs therefore appear to be an effective and useful tool to predict peptide epitopes of proteins associated with cancer.     

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.     

Roles of the hinge region and the DNA binding domain of the bovine papillomavirus type 1 E2 protein in initiation of DNA replication

The bovine papillomavirus (BPV-1) E2 protein is the regulator of extrachromosomal replication of papillomaviruses. The mutants with C-terminal truncations and in-frame internal deletions were constructed to study the role of structural domains of E2 in the initiation of DNA replication. We show that the replication initiation function of E2 is absolutely dependent on the ability of the protein to bind to DNA. Our study also confirms the borders of the functional domains of the E2 protein; residues 1-192 form the activation domain and residues 310-410 the DNA binding-dimerization domain. Some critical length and flexibility, but not the particular amino acid sequence between these two functional domains is required for the activity of the protein to support replication. The hinge region, including the major phosphorylation sites of E2, is also dispensable for the mediation of attachment of the BPV1 genome to the mitotic chromosomes.     

Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions

The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein's ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5.     

Expression of platelet-derived growth factor-beta receptor and bovine papillomavirus E5 and E7 oncoproteins in equine sarcoid

Equine sarcoids are benign fibroblastic skin tumours that are recognized throughout the world. Infection with bovine papillomavirus (BPV) types 1 and 2 has been implicated as a major factor in disease development; however, the cellular mechanisms underlying fibroblast transformation remain poorly defined. The present study further characterizes aspects of the association with BPV in 15 equine sarcoids. BPV DNA was demonstrated in 12/15 tumours collected from different areas of Italy. Nine of these 12 tumours expressed the BPV oncoproteins E5 and E7, but these oncoproteins were not expressed by normal equine cells. The BPV E5 protein is known to bind to the platelet-derived growth factor-beta receptor (PDGF-betaR) and this molecule was expressed by 11 of the 12 sarcoids in which E5 was demonstrated. These findings add further weight to the theory that BPV and the PDGF-betaR may have a role in the pathogenesis of this disease.     

Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions.     

The interface between MyBP-C and myosin: site-directed mutagenesis of the CX myosin-binding domain of MyBP-C

Myosin-binding protein-C (MyBP-C or C-protein) is a ca. 130 kDa protein present in the thick filaments of all vertebrate striated muscle. The protein contains ten domains, each of ca. 90–100 amino acids; seven are members of the IgI family of proteins, three of the fibronectin type III family. The motifs are arranged in the following order (from N- to C-terminus): Ig-Ig-Ig-Ig-Ig-Fn-Fn-Ig-Fn-Ig. The C-terminal Ig motif (domain X or CX) contains its light meromyosin-binding site. A recombinant form of CX, beginning at Met-1027, exhibits saturable binding to myosin with an affinity comparable to the C-terminal 13 kDa chymotryptic fragment of native MyBP-C. To identify the surface in CX involved in its interaction with myosin, nine site-directed mutants (R37E, K43E, N49D, E52R, D56K, R73E, R74E, G80D and R103E) were constructed. Using a new assay for assessing the binding of CX with the light meromyosin (LMM) portion of myosin, we demonstrate that recombinant CX, just as the full-length protein, is able to facilitate LMM polymerization. Moreover, we show that residues Arg-37, Glu-52, Asp-56, Arg-73, and Arg-74 are involved in this interaction with the myosin rod. All of these amino acids interact with negatively charged residues of LMM, since the mutants R37E, R73E and R74E are unable to bind myosin, whereas E52R and D56K bind myosin with higher affinity than wild-type CX. Residues Lys-43 and Arg-103 show a small but significant influence on the binding reaction; residues Asn-49 and Gly-80 seem not to be involved in this interaction. Based on these data, a model is proposed for the interaction between MyBP-C CX and myosin filaments. In this model, CX interacts with four molecules of LMM at four different sites of the binding protein, thus explaining the effects of MyBP-C on the critical concentration of myosin polymerization.     

Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2, -3, -4 and -10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features.