Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.     

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.     

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.     

Human Papillomavirus Oncoproteins E6 and E7 Independently Abrogate the Mitotic Spindle Checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G₁/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G₂ checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G₂ checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.     

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background

Human Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.

Methodology/Principal Findings

By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.

Conclusions/Significance

This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.     

Functional Similarity between E6 Proteins of Cutaneous Human Papillomaviruses and the Adenovirus E1A Tumor-Restraining Module

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.     

Pericentromeric Regions Are Refractory To Prompt Repair after Replication Stress-Induced Breakage in HPV16 E6E7-Expressing Epithelial Cells

Chromosomal instability is the major form of genomic instability in cancer cells. Amongst various forms of chromosomal instability, pericentromeric or centromeric instability remains particularly poorly understood. In the present study, we found that pericentromeric instability, evidenced by dynamic formation of pericentromeric or centromeric rearrangements, breaks, deletions or iso-chromosomes, was a general phenomenon in human cells immortalized by expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7). In particular, for the first time, we surprisingly found a dramatic increase in the proportion of pericentromeric chromosomal aberrations relative to total aberrations in HPV16 E6E7-expressing cells 72 h after release from aphidicolin (APH)-induced replication stress, with pericentromeric chromosomal aberrations becoming the predominant type of structural aberrations (∼70% of total aberrations). In contrast, pericentromeric aberrations accounted for only about 20% of total aberrations in cells at the end of APH treatment. This increase in relative proportion of pericentromeric aberrations after release from APH treatment revealed that pericentromeric breaks induced by replication stress are refractory to prompt repair in HPV16 E6E7-expressing epithelial cells. Telomerase-immortalized epithelial cells without HPV16 E6E7 expression did not exhibit such preferential pericentromeric instability after release from APH treatment. Cancer development is often associated with replication stress. Since HPV16 E6 and E7 inactivate p53 and Rb, and p53 and Rb pathway defects are common in cancer, our finding that pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing cells may shed light on mechanism of general pericentromeric instability in cancer.     

Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.     

Adenovirus E1A,not human papillomavirus E7, sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-alpha-dependent mechanisms despite up-regulation of 70-kDa heat shock protein

Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-alpha, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-alpha, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-alpha. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-alpha and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-alpha, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-alpha, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-alpha, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-alpha-dependent mechanisms.     

Cutaneous human papillomavirus type 38 E7 regulates actin cytoskeleton structure for increasing cell proliferation through CK2 and the eukaryotic elongation factor 1A

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.