High-risk human papillomavirus E6 protein has two distinct binding sites within p53, of which only one determines degradation.

Human papillomavirus (HPV) E6 protein can inactivate tumor suppressor p53 by inducing its degradation. We now find that high-risk HPV E6 binds to p53 at two distinct sites; one is within the core structure of p53, and another is at the C terminus of p53. Binding to the core of p53 is required for E6-mediated degradation, as shown by deletion analysis and the properties of a point mutant at residue 135. Both low- and high-risk HPV E6 can bind to a C-terminal region of p53, but these interactions do not induce degradation. These results resolve previous seemingly contradictory findings that attributed the distinctive functional properties of high- and low-risk E6 proteins to either a difference in their abilities to associate with p53 or a difference in their N-terminal structures.     

Genomic diversity and evolution of papillomaviruses in rhesus monkeys.

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.     

Degradation of p53 by Human Alphapapillomavirus E6 Proteins Shows a Stronger Correlation with Phylogeny than Oncogenicity

Background

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.

Principal Findings

We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.

Conclusions

These data suggest that the alpha-HPV E6 proteins'' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.     

Involvement of nuclear export in human papillomavirus type 18 E6-mediated ubiquitination and degradation of p53

The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.     

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.     

Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins.

The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.     

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.     

Alpha1,4-fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently

In the animal kingdom the enzymes that catalyze the formation of alpha1,4 fucosylated-glycoconjugates are known only in apes (chimpanzee) and humans. They are encoded by FUT3 and FUT5 genes, two members of the Lewis FUT5-FUT3-FUT6 gene cluster, which had originated by duplications of an alpha3 ancestor gene. In order to explore more precisely the emergence of the alpha1,4 fucosylation, new Lewis-like fucosyltransferase genes were studied in species belonging to the three main primate groups. Two Lewis-like genes were found in brown and ruffed lemurs (prosimians) as well as in squirrel monkey (New World monkey). In the latter, one gene encodes an enzyme which transfers fucose only in alpha1,3 linkage, whereas the other is a pseudogene. Three genes homologous to chimpanzee and human Lewis genes were identified in rhesus macaque (Old World monkey), and only one encodes an alpha3/4-fucosyltransferase. The ability of new primate enzymes to transfer fucose in alpha1,3 or alpha1,3/4 linkage confirms that the amino acid R or W in the acceptor-binding motif "HH(R/W)(D/E)" is required for the type 1/type 2 acceptor specificity. Expression of rhesus macaque genes proved that fucose transfer in alpha1,4 linkage is not restricted to the hominoid family and may be extended to other Old World monkeys. Moreover, the presence of only one enzyme supporting the alpha1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution.     

Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes.

The E6 proteins of the high-risk human papillomaviruses (HPVs) have been shown to form a complex with and induce the degradation of human p53 in vitro. To determine whether p53 is degraded more rapidly in cells expressing E6 in vivo, the half-life of p53 was determined by pulse-chase analysis in early-passage normal human keratinocytes and fibroblasts, human keratinocytes immortalized with HPV type 16 (HPV16) E6 plus E7, and nonimmortal keratinocytes transfected with E6. The results of these experiments indicate that (i) the half-life of newly synthesized p53 is relatively long (4 h) in early-passage human keratinocytes and fibroblasts but short in keratinocytes expressing E6 (15 to 30 min), (ii) a similar increased rate of p53 degradation was measured in lines immortalized with HPV16 E6 plus E7 and senescent cells expressing E6, indicating that this increase is not simply the result of selection in the immortalized lines, and (iii) very low levels of expression of E6 result in a greatly decreased half-life of p53, suggesting that E6 acts in a catalytic manner.     

The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the 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 (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.     

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.     

Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.     

Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.     

Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization,Biophysical Characterization,and Phylogenetic Analysis

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.Papillomaviruses (PVs) are nonenveloped, epitheliotropic, double-stranded DNA viruses that cause a variety of diseases in a multitude of hosts. Based on available whole-genome sequences and subgenomic amplicons, more than 200 human and over 55 nonhuman mammalian PV types have been described (7, 34, 35, 37, 38). To date, two avian PV types have been characterized (37, 38).The genomic organizations of the PVs are remarkably similar. The genome is ca. 8 kb in length and comprises an upstream regulatory region (URR), the early genes (E1, E2, E4, E6, and E7), and the late genes that encode the capsid proteins (L1 and L2). Although most PVs code for these seven open reading frames (ORFs), only the URR, the replicative proteins E1 and E2 (and possibly the E4 gene), and the capsid proteins L1 and L2 are strictly conserved in all PVs (11).Upon infection of the stratified squamous epithelia, PV gene expression is linked to the differentiation state of the infected epithelium cells. The expression of early PV proteins, in particular E6 and E7, primes the proliferation of the infected epithelium. This proliferation, which is absolutely required for viral replication, may become malignant depending on the PV strain considered. Several “high-risk” mucosal human PV (HPV) strains (predominantly HPV type 16 [HPV-16], HPV-18, and HPV-45) have been shown to be responsible for cervical cancer (19).The ability of PVs to induce proliferation of the infected cells has been attributed mainly to two small “oncoproteins,” E6 and E7. In genital high-risk HPVs, these proteins play a prominent role in cell immortalization and transformation (31). In most mammalian PVs, E6 is a small protein of about 150 amino acids, with two conserved N- and C-terminal zinc-binding domains, E6N and E6C, respectively (12). The solution structure of the HPV-16 E6C domain was recently determined (23). The sequence alignments pointed to a structural similarity between the E6C and E6N domains, suggesting that a single-domain protein possessing the same fold might have once existed. Earlier phylogenetic studies had suggested that gene duplication may have given rise to the current double-domain E6 proteins (5). Interestingly, although the E6 ORF has been found in most mammalian PVs (with the exception of bovine papillomavirus type 3 [BPV-3], BPV-4, BPV-6, HPV-101, and HPV-103 [3, 7]), it was not detected in the two avian PVs previously sequenced (37, 38).In this study, we present the full sequence of the genome of a novel PV from a francolin bird (Francolinus leucoscepus PV type 1 [FlPV-1]) and compare it to the two other avian PV genomes known to date (Psittacus erithacus PV [PePV] and Fringilla coelebs PV [FPV]). In light of recent structural data, we compare the unusual avian E6 and E7 ORFs to their mammalian orthologs. We describe the expression and purification of recombinant avian PV E6 and E7 proteins, their biophysical characterization, and cellular localization. Finally, we use phylogenetic techniques to investigate the evolutionary history of the E6 protein family.     

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.     

Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.