Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation

The E6 proteins from the high-risk human papillomavirus (HPV) types have previously been shown to target a number of PDZ domain-containing proteins for proteasome-mediated degradation. These include the hDlg tumour suppressor and the MAGI-1 protein. In this study we show that high-risk HPV E6 proteins also target the related MAGI-2 and MAGI-3 proteins for degradation. Moreover, we show that the interaction is specific to one PDZ domain, and that co-expression of this domain can protect each of the full-length MAGI proteins from E6-mediated degradation. These data provide clear indicators for the potential design of compounds that could specifically inhibit the interaction of oncogenic HPV E6 proteins with an important class of target proteins.     

The role of human papillomavirus oncoproteins E6 and E7 in apoptosis

The oncogenic potential of 'high risk' human papillomaviruses can be mainly attributed to two small proteins called E6 and E7. Even these oncoproteins have a low molecular size, they are highly promiscuous and are capable to interact with a whole variety of host cellular regulator proteins to elicit cellular immortalization and ultimately complete malignant transformation. To avoid reiterations in summarizing the biochemical and molecular biological properties of E6/E7 in terms of their influence on cell cycle control, the present review is mainly an attempt to describe some regulatory principles by which human papillomavirus (HPV) oncoproteins can interfere with apoptosis in order to escape immunological surveillance during progression to cervical cancer. The models derived from these basic cellular and molecular studies are relevant to our understanding of HPV-induced carcinogenesis. Conversely, experimental procedures aimed at relieving apoptosis resistance, can facilitate the eradication of immunologically suspicious cells and may prevent the accumulation of cervical intraepithelial cell abnormalities in future prophylactic or therapeutic approaches.     

Selective PDZ protein-dependent stimulation of phosphatidylinositol 3-kinase by the adenovirus E4-ORF1 oncoprotein

While PDZ domain-containing proteins represent cellular targets for several different viral oncoproteins, including human papillomavirus E6, human T-cell leukemia virus type 1 Tax, and human adenovirus E4-ORF1, the functional consequences for such interactions have not been elucidated. Here we report that, at the plasma membrane of cells, the adenovirus E4-ORF1 oncoprotein selectively and potently stimulates phosphatidylinositol 3-kinase (PI3K), triggering a downstream cascade of events that includes activation of both protein kinase B and p70S6-kinase. This activity of E4-ORF1 could be abrogated by overexpression of its PDZ-protein targets or by disruption of its PDZ domain-binding motif, which was shown to mediate complex formation between E4-ORF1 and PDZ proteins at the plasma membrane of cells. Furthermore, E4-ORF1 mutants unable to activate the PI3K pathway failed to transform cells in culture or to promote tumors in animals, and drugs that block either PI3K or p70S6-kinase inhibited E4-ORF1-induced transformation of cells. From these results, we propose that the transforming and tumorigenic potentials of the adenovirus E4-ORF1 oncoprotein depend on its capacity to activate PI3K through a novel PDZ protein-dependent mechanism of action.     

Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.     

DNA vaccines against the human papillomavirus type 16 E6 or E7 oncoproteins

DNA vaccines expressing the E6 or E7 oncoproteins of human papilloma virus type 16 (HPV-16) in either their wild-type form or fused to sequences that affect intracellular trafficking were tested for induction of protective immunity against tumor cell challenge in two models based on BALB/c and C57Bl/6 mice. The DNA vaccines to E7 gave uniformly disappointing results, while the DNA vaccine that expressed E6 linked to a viral leader sequence protected BALB/c mice against tumor cell challenge given before or after vaccination. The efficacy of this vaccine could be enhanced by a DNA vector prime/viral vector boost regimen. In contrast, priming of mice with the DNA vaccines to E7 reduced the efficacy of a viral vector expressing the same antigen.     

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in patients with head-and-neck squamous-cell carcinoma

Human papillomaviruses (HPVs) have been recognized as an essential pathogenic factor in anogenital cancer. HPV DNA has also been found in a subgroup of head-and-neck squamous-cell carcinomas (HNSCCs), and a causative role of the virus in the development of these tumors has been suggested by the concomitant inactivation of the tumor-suppressor protein pRb. Using 4 second-generation ELISAs, we found antibodies against at least 1 of the oncoproteins E6 and E7 of the high-risk HPV types 16 and 18 in 11 of 92 sera (12%) taken from HNSCC patients at or near diagnosis, in 1 of 52 sera (2%) taken from HNSCC patients >6 months after diagnosis and in 10 of 288 sera (3. 5%) taken from sex- and age-matched healthy control individuals of the normal population. In 11 of the 12 seropositive HNSCC cases, antibodies were directed against HPV16 proteins. In patients, the HPV16 antibodies were mostly of high titer, and in 6 cases, antibodies against both HPV16 oncoproteins were present. Seven of the 8 HPV16 antibody-positive sera from the control group were of low titer, and none of the 10 antibody-positive sera reacted with both oncoproteins of the same HPV type. The HPV type of the antigens detected by the antibodies in HNSCC patients correlated well with that of the HPV DNA found in the tumor. Of 19 patients known to have HPV16 DNA-positive tumors, 7 (37%) also had HPV16 E6 and/or E7 antibodies. Our finding suggests that the antibodies were formed in an immune response against HPV E6 and E7 proteins expressed in the HNSCC and thus strongly supports the concept of a biologically active role of HPV in the development of a subgroup of HNSCC.     

The human papillomavirus type 16 E6 and E7 oncoproteins independently induce numerical and structural chromosome instability

The development of genomic instability is a hallmark of high-risk human papillomavirus (HPV) associated cervical carcinogenesis. We have previously shown that the HPV-16 E7 oncoprotein rapidly subverts mitotic fidelity by inducing abnormal centrosome numbers and multipolar mitotic spindles. Here we report that expression of HPV-16 E6 and E7 independently results in various mitotic abnormalities. HPV-16 E6 and E7 were each associated with unaligned or lagging chromosomal material, indicating relaxation of spindle checkpoint control. Moreover, by overwhelming checkpoint control mechanisms that may prevent cells with multiple spindle poles to enter anaphase, expression of HPV-16 E6 and E7 leads to a small but significant number of cells with altered polarity at later stages of the cell division process. In addition to changes that have the potential to give rise to numerical chromosome imbalances, we discovered that expression of HPV-16 E7 could trigger anaphase bridge formation to an extent similar to that of high-risk HPV E6. Anaphase bridges typically develop after chromosomal breaks and alterations of chromosomal structure. Further investigation of mechanisms by which HPV-16 E6 and E7 contribute to the destabilization of the host cell genome revealed that both high-risk HPV oncoproteins induce DNA damage. Moreover, expression of HPV-16 E7 was associated with an increased number of cells exhibiting nuclear foci of phosphorylated histone H2AX as well as activation of cell cycle checkpoints triggered by DNA repair. Our results therefore suggest that HPV oncoproteins are a source for both numerical and structural chromosome instability during HPV-associated carcinogenesis.     

Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells

Infection of cervical keratinocytes by high-risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP-1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high-risk HPV, individually and together, suppress MCP-1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP-10, IL-8 and RANTES, were less affected. Furthermore, 4 of 6 HPV-positive cervical carcinoma cell lines did not express MCP-1. Our data indicate that suppression of MCP-1 expression is part of the program of high-risk HPV E6/E7-induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP-1 expression by infected keratinocytes, which would stimulate an immune attack on HPV-transformed cells, is suppressed for invasive cervical cancer to appear.     

高危型人乳头瘤病毒癌基因E6和E7调节细胞作用靶点的研究进展

高危型人乳头瘤病毒(human papillomavirus,HPV)的感染与多种人类癌症密切相关,其中最典型的是宫颈癌.高危型HPV最重要的两个病毒癌基因为E6和E7,病毒基因组整合到宿主基因组是病毒癌基因E6和E7实现持续表达的一种方式.HPV癌基因E6和E7能够靶向宿主细胞途径,通过这些相互作用,导致HPV发挥其...     

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in cervical-carcinoma patients from Russia

Certain human papillomaviruses (HPV), mainly types 16 and 18, have been widely recognized as an essential etiologic factor for the development of carcinoma of the uterine cervix. The early HPV proteins E6 and E7 are consistently expressed in the tumor cells, and cervical-carcinoma patients can develop antibodies against these oncoproteins. For cervical-carcinoma patients from Eastern Europe and Russia, detailed information on HPV DNA prevalence and HPV-specific immune responses is limited. The presence of HPV DNA in 128 Russian cervical-carcinoma tissues was determined: HPV16 DNA was found in 78% of the cases, HPV18 DNA in 14%, and no HPV-DNA in 10%. Using 4 recently developed sensitive and highly specific second-generation enzyme-linked immunosorbent assays, we also analyzed the prevalence of antibodies against HPV16 and -18 E6 and E7 proteins in sera from 95 cervical-carcinoma patients, from 61 female patients with non-HPV-associated tumors and from 83 female healthy controls. The strong association of E6 and/or E7 antibodies with cervical carcinoma was confirmed, with 36% seropositives in this group against only 2% in the control groups. The detected antibodies are highly HPV-type-specific since all 26 HPV16-E6- or -E7-antibody-positive patients had HPV16 DNA in their tumor and 6 out of the 8 HPV18-antibody-positive patients had HPV18 DNA. Antibody responses to HPV16 E6 and E7 appear to be dependent on clinical stage of the disease, with 21% seropositives found in FIGO stage I, 42% in stage II and 53% in stage III. Antibody response to HPV16 E6 is more frequent than to E7, especially in early stages.     

Ubiquitin-independent degradation of p53 mediated by high-risk human papillomavirus protein E6

In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.     

Down-regulation of MHC class I by bovine papillomavirus E5 oncoproteins.

The papillomavirus E5 protein is localized in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of the host cell. Transformed bovine fibroblasts expressing bovine papillomavirus (BPV) E5 are highly vacuolated and have a much enlarged, distorted and fragmented GA. Major histocompatibility complex class I (MHC I) is processed and transported to the cell surface through the GA. Given the cellular localization of E5 in the GA and the morphologically abnormal GA, we investigated the expression of MHC I in cells transformed by E5 from BPV-1 and BPV-4. Two cell lines were used: bovine cells that also express E6, E7 and activated ras, and NIH3T3 cells that express only E5. In addition, PalF cells acutely infected with a recombinant retrovirus expressing E5 were also examined. In contrast to non-transformed normal cells, or transformed cells expressing other papillomavirus proteins, cells expressing E5 do not express MHC I on their surface, but retain it intracellularly, independently of the presence of other viral or cellular oncogenes, or of whether the cells are long-term transformants or acutely infected. We conclude that expression of E5 prevents expression of MHC I to the cell surface and causes its retention within the cell. In addition, lower amounts of total MHC I heavy chain and of heavy chain RNA are detected in E5-transformed cells than in control cells. As surface expression of another glycosylated membrane protein, the transferrin receptor, is not affected, it appears that E5 targets MHC I with at least a degree of specificity. In papillomavirus lesions this effect would have important implications for antigen presentation by, and immunosurveillance of, virally infected cells.     

Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors

Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro- and antiangiogenic factors by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic factors and/or increased levels of proangiogenic factors. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and vascular endothelial growth factor (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF 121, 145, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of growth and spread.     

Interactions of the human papillomavirus E7 protein with cell cycle regulators

Human papillomaviruses (HPVs) critically depend on the cellular machinery for the replication of their genome. Viral replication is restricted to the differentiated strata of the skin that are normally growth arrested. Hence, the HPVs have developed strategies to subvert cellular growth regulatory pathways and are able to uncouple cellular proliferation and differentiation. The HPV E7 protein can overcome the activity of some cyclin-dependent kinase inhibitors, associate with cyclin/cyclin dependent kinase complexes and bind to and destabilize the retinoblastoma tumor suppressor protein. These biological activities contribute to the carcinogenic potential of the high risk HPV E7 proteins which are consistently expressed in HPV-positive cervical carcinomas.     

Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase

High-risk human papilloma viruses are known to be associated with cervical cancers. We have reported previously that the high-risk human papillomavirus (HPV) E6 oncoprotein interacts with E6TP1, a novel Rap GTPase-activating protein (RapGAP). Similar to p53 tumor suppressor protein, the high-risk HPV E6 oncoproteins target E6TP1 for degradation. The HPV16 E6-induced degradation of E6TP1 strongly correlates with its ability to immortalize human mammary epithelial cells. In this study, we used treatment with a proteasome inhibitor MG132, analysis in CHO-ts20 cells with a thermolabile ubiquitin-activating enzyme, and direct detection of ubiquitin-modified E6TP1 to demonstrate that E6TP1 is targeted for degradation by the ubiquitin-proteasome pathway both in the presence and in the absence of E6. Using deletion mutants of E6TP1, we mapped the region required and sufficient for E6 binding to COOH-terminal 40 amino acid residues and showed this region to be necessary for E6-dependent degradation of E6TP1. Furthermore, the E6-binding region of E6TP1 complexes with E6AP via E6. Importantly, the purified E6AP enhanced the ubiquitination and degradation of E6TP1 in the presence of E6 in vitro. Additionally, the expression of a dominant-negative E6AP mutant (C833A) in cells inhibited the E6-induced degradation of E6TP1. These findings demonstrate that the E6-induced decrease in the levels of E6TP1 protein involves the E6AP-mediated ubiquitination followed by proteasome-dependent degradation.     

人乳头瘤病毒E6与宫颈癌

宫颈癌的病因与持续感染人乳头瘤病毒(human papillomavirus,HPV)相关.最近的研究表明,HPV E6在宫颈癌的发生过程中发挥重要作用.本文就HPV E6宫颈癌的发生机制及HPV E6蛋白的表达与宫颈癌的临床意义进行综述.