The human papillomavirus (HPV) 16 E2 protein induces apoptosis in the absence of other HPV proteins and via a p53-dependent pathway

The human papillomavirus (HPV) E2 protein regulates viral gene expression and is also required for viral replication. HPV-transformed cells often contain chromosomally integrated copies of the HPV genome in which the viral E2 gene is disrupted. We have shown previously that re-expression of the HPV 16 E2 protein in HPV 16-transformed cells results in cell death via apoptosis. Here we show that the HPV 16 E2 protein can induce apoptosis in both HPV-transformed and non-HPV-transformed cell lines. E2-induced apoptosis is abrogated by a trans-dominant negative mutant of p53 or by overexpression of the HPV 16 E6 protein, but is increased by overexpression of wild-type p53. We show that mutations that block the DNA binding activity of E2 do not impair the ability of this protein to induce apoptosis. In contrast, removal of both N-terminal domains from the E2 dimer completely blocks E2-induced cell death. Heterodimers formed between wild-type E2 and N-terminally deleted E2 proteins also fail to induce cell death. Our data suggest that neither the DNA binding activity of E2 nor other HPV proteins are required for the induction of apoptosis by E2 and that E2-induced cell death occurs via a p53-dependent pathway.     

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.     

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.     

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.     

HPV16 E6通过阻碍ING4对p53作用而抑制细胞凋亡的实验研究

通过HPV16 E6干扰ING4对p53作用的实验研究,探讨HPV16 E6新的致癌机制。采用转染及免疫共沉淀实验证明HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白乙酰化的作用;将表达p53、ING4和p53报告基因与HPV16 E6或其突变体的质粒共转染p53蛋白阴性的SaoS2细胞系,荧光素酶报告基因检测HPV16 E6抑制ING4对p53基因在转录水平的影响;并采用细胞集落形成实验检测HPV16 E6对ING4所诱导p53途径所致细胞凋亡的抑制。HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白Lys-382的乙酰化;HPV16 E6减弱ING4在转录水平对p53基因的调控,HPV16 E6抑制ING4诱导的p53途径介导的细胞凋亡,且所有这些作用不依赖p53蛋白的降解。HPV16 E6阻碍ING4对p53的作用而抑制细胞凋亡可能是其引起癌变的途径之一。     

Comparison of the structure and DNA-binding properties of the E2 proteins from an oncogenic and a non-oncogenic human papillomavirus

Human papillomaviruses (HPVS) that infect the genital tract can be divided into two groups: high-risk HPV types, such as HPV 16 and HPV 18, are associated with cancer, low-risk HPV types, such as HPV 6, are associated with benign warts. In both high-risk and low-risk HPV types, the papillomavirus E2 protein binds to four sites within the viral long control region (LCR) and regulates viral gene expression. Here, we present the crystal structure of the minimal DNA-binding domain (DBD) from the HPV 6 E2 protein. We show that the HPV 6 E2 DBD is structurally more similar to the HPV 18 and bovine papillomavirus type 1 (BPV1) E2 proteins than it is to the HPV 16 E2 protein. Using gel retardation assays, we show that the hierarchy of E2 sites within the HPV 16 and HPV 6 LCRs are different. However, despite these differences in structure and site preference, both the HPV 16 and 6 E2 DBDs recognise an extended version of the consensus E2 binding site derived from studies of the BPV1 E2 protein. In both cases, the preferred binding site is 5'AACCGN(4)CGGTT3', where the additional flanking base-pairs are in bold and N(4) represents a four base-pair central spacer. Both of these HPV proteins bind preferentially to E2 sites that contain an A:T-rich central spacer. We show that the preference for an A:T-rich central spacer is due, at least in part, to the need to adopt a DNA conformation that facilitates protein contacts with the flanking base-pairs.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.     

High-Risk But Not Low-Risk HPV E2 Proteins Bind to the APC Activators Cdh1 and Cdc20 and Cause Genomic Instability

Human papillomaviruses (HPVs) from the high-risk group are associated with cervicalcancer, in contrast to HPVs from the low-risk group which are associated with benignlesions. Here, we show that high-risk, but not low-risk HPV E2 proteins, promote amitotic block, often followed by metaphase-specific apoptosis, and which is independentof the viral oncogenes E6 and E7. High-risk HPV E2-expressing cells also showpolyploidy, chromosomal mis-segregation and centrosome amplification leading togenomic instability. We link these defects to a specific and unusually strong interactionbetween high-risk E2 and both Cdc20 and Cdh1, two activators of the AnaphasePromoting Complex (APC), abnormal localization of Cdh1, and accumulation of APCsubstrates like cyclin B, in vivo. The finding that high-risk, but not low-risk HPV E2proteins, induce genomic instability, raises the intriguing possibility that E2 proteinsplay a role in the oncogenic potential of high-risk papillomaviruses.     

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Human Papillomavirus E7 Protein Deregulates Mitosis via an Association with Nuclear Mitotic Apparatus Protein 1

We previously observed that high-risk human papillomavirus type 16 (HPV16) E7 expression leads to the delocalization of dynein from mitotic spindles (C. L. Nguyen, M. E. McLaughlin-Drubin, and K. Munger, Cancer Res. 68:8715-8722, 2008). Here, we show that HPV16 E7 associates with nuclear mitotic apparatus protein 1 (NuMA) and that NuMA binding and the ability to induce dynein delocalization map to similar carboxyl-terminal sequences of E7. Additionally, we show that the delocalization of dynein from mitotic spindles by HPV16 E7 and the interaction between HPV16 E7 and NuMA correlate with the induction of defects in chromosome alignment during prometaphase even in cells with normal centrosome numbers. Furthermore, low-risk HPV6b and HPV11 E7s also associate with NuMA and also induce a similar mitotic defect. It is possible that the disruption of mitotic events by HPV E7, via targeting of the NuMA/dynein complex and potentially other NuMA-containing complexes, contributes to viral maintenance and propagation potentially through abrogating the differentiation program of the infected epithelium. Furthermore, in concert with activities specific to high-risk HPV E6 and E7, such as the inactivation of the p53 and pRB tumor suppressors, respectively, the disruption of the NuMA/dynein network may result in mitotic errors that would make an infected cell more prone to the accumulation of aneuploidy even in the absence of supernumerary centrosomes.     

Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification.     

Roles of the E6 and E7 proteins in the life cycle of low-risk human papillomavirus type 11

Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.     

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)