Mutant E1 Research Articles

Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF( E6-EGFP ) cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF( E6-EGFP ) cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF( E6-EGFP ) cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF( E6-EGFP ) cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF( E6-EGFP ) cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.

Brd4 Is Required for E2-Mediated Transcriptional Activation but Not Genome Partitioning of All Papillomaviruses

Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes.

A Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G436WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G436WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.

The Human Papillomavirus Type 16 E7 Protein Binds Human Interferon Regulatory Factor-9 via a Novel PEST Domain Required for Transformation

It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7-IRF-9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

Gene expression profiles reveal an upregulation of E2F and downregulation of interferon targets by HPV18 but no changes between keratinocytes with integrated or episomal viral genomes

Persistent infections with human papillomaviruses type 18 can result in the development of cervical cancer. HPV18 genomes persist extrachromosomally in low-grade and precancerous lesions but are always integrated in cervical cancers, and this might contribute to the progression of HPV18-induced lesions. To address whether integration induces additional changes in host cells, several keratinocyte lines with wild type and replication-deficient E1 mutant HPV18 (E1C-TTL) genomes were analyzed with high density oligonucleotide arrays. In comparison to normal keratinocytes, wild type and integrated E1C-TTL HPV18 genomes deregulate the expression of 280 annotated genes. However, the comparison of wild type with E1C-TTL cell lines did not reveal any significant differences, indicating that neither the loss of E1 nor viral integration induces additional gene expression changes in low passage HPV18-positive keratinocytes. Half of the deregulated genes have been described as targets of the p16/Rb/E2F, p53, interferon or NFκB pathways consistent with the functions ascribed to the viral E6 and E7 oncoproteins, but the other half can currently not be ascribed to certain pathways.

Second-site revertants of a Semliki Forest virus fusion-block mutation reveal the dynamics of a class II membrane fusion protein.

The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.

Repression of HPV16 early region transcription by the E2 protein

HPV16 DNA is often integrated in cancers, disrupting the E1 or E2 genes. E2 can repress the E6/E7 promoter, but other models have been proposed to explain why integration promotes malignant progression. E1 and E2 are required for viral replication, and so genetic analysis of their role in transcriptional regulation is complex. Therefore, we developed an extrachromosomal vector containing HPV16 to undertake a genetic analysis of the E1 and E2 genes. We demonstrate that the E2 protein is primarily a transcriptional repressor when expressed from the virus. Furthermore, repression requires both the transactivation function of E2 and specific binding of E2 to the LCR. We find no evidence that the E1 protein directly modulates HPV16 gene expression. However, certain E1 mutations modulated transcription indirectly by altering splicing of E2 mRNA species. These data provide important insight into which E1 and E2 functions are optimal targets for anti-viral therapies.

The 20S proteasome processes NF-κB1 p105 into p50 in a translation-independent manner

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.

Replication Properties of Human Adenovirus In Vivo and in Cultures of Primary Cells from Different Animal Species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Effects of human papillomavirus type 16 oncoproteins on survivin gene expression

Survivin has recently been identified as a novel member of the inhibitor of apoptosis (IAP) gene family. The product of this gene not only suppresses apoptosis but also controls cell division. Survivin is undetectable in most terminally differentiated normal tissues but is expressed in embryonic and fetal organs and is present in most malignant tumours. Human papillomaviruses (HPV) are thought to play an important role in the development of cervical cancer. By interfering in the cell cycle, the viral oncoproteins (E6 and E7) can induce the immortalization of the host cell. The transcriptional effects of the HPV-16 E6 and E7 proteins on the survivin promoter in transiently transfected cell lines using luciferase tests were examined. HPV-16 E6, but not E7, was found to significantly transactivate the survivin promoter. Experiments performed in different cancer cell lines and with different E6 mutants indicated that the effect of E6 on the survivin promoter is largely dependent on p53 status. In accordance with this, the p53 tumour suppressor protein downregulated the expression of survivin. As E6 is able to interact with p53 and induces its ubiquitin-dependent degradation, it appears that the transactivation effect of E6 on survivin is mediated by the p53 degradation pathway. Transduction of HPV-16 E6 and E7 into human embryonic fibroblast cells showed that the HPV oncoproteins can upregulate endogenous survivin mRNA. Importantly, cell cycle synchronization experiments showed that the effect of HPV-16 E6 on survivin transcription is independent of the cell cycle.

Requirement of E7 oncoprotein for viability of HeLa cells

Most human papillomavirus (HPV)-positive cervical cancers contain integrated copies of the viral genome in their chromosomes and express the viral oncoproteins E6 and E7. A virus-encoded transcription factor, E2, is known to repress E6/E7 expression in HPV-positive cancer cells, leading to growth inhibition, which indicates that E6/E7 is required for the survival of the cells. We found that the E2-mediated growth inhibition of HeLa cells, an HPV18-positive cancer cell line, was coupled with a reduction in telomerase activity, an effect which was rescued by the complementation of E7 expression, but not E6 expression, indicating that the cell viability and the telomerase activity in HeLa cells are maintained by an E7-associated function. Analysis of E7 mutants suggested that the binding to the pRB family of pocket proteins was involved in the ability of E7 to rescue the growth potential and telomerase activity inhibited by E2 expression. We also showed that the telomerase activity upregulated by E7 expression was determined by the hTERT promoter activity, and that c-Myc upregulation caused by pRB inactivation could account for the promoter activity. The activation of p53 and consequent accumulation of p21Cip1, which were triggered by the downregulation of E6, appeared not to be essential for the E2-mediated growth arrest.

A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-d-xylulose 5-phosphate synthase

The 2-C-methyl-d-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.

In vivo Imaging of Adenovirus Transduction and Enhanced Therapeutic Efficacy of Combination Therapy with Conditionally Replicating Adenovirus and Adenovirus-p27

Gene therapy is hampered by poor gene transfer to the tumor mass. We previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 (delta24RGD) and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Mutant E1 expressed by delta24RGD enables the replication of replication-defective adenoviruses in tumors when cancer cells are co-infected with both viruses. In this study, gene transfer rates in xenografts tumors were monitored by bioluminescence in cells infected with the replication-defective adenovirus-luciferase (ad-luc). Tumor masses treated with CRAD + ad-luc showed dramatically stronger and more prolonged luciferase expression than ad-luc-treated tumors and this expression spread through the entire tumor mass without significant systemic spread. Transduction with CRAD + replication-defective adenovirus-p27 increased the expression of p27 by 24-fold versus transduction with ad-p27 alone. Treatment of a lung cancer cell line and of established lung cancer xenografts with CRAD + adenovirus-p27 also induced stronger growth suppression than treatment with either virus alone. These findings confirm the selective replication of E1-deleted adenovirus containing a therapeutic gene due to the presence of mutant E1 produced by delta24RGD in tumors. Moreover, this replication increased the therapeutic gene transfer rate and enhanced its antitumor effects.

Involvement of a cellular ubiquitin-protein ligase E6AP in the ubiquitin-mediated degradation of extensive substrates of high-risk human papillomavirus E6

Human scribble (hScrib), which was identified as substrate of human papillomavirus (HPV) E6 for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP, is a human homolog of Drosophila neoplastic tumor suppressor scribble, in which mutation causes loss of polarity and overgrowth of epithelia. Drosophila discs large (Dlg) is one of neoplastic tumor suppressors, which genetically links to scribble. E6 also targets human Dlg (hDlg) for ubiquitin-mediated degradation. Ubiquitin-protein ligase involved in this process has not been identified thus far. Here we investigated mechanism underlying degradation of three target proteins of E6, hScrib, hDlg, and p53 by using eighteen HPV 16 E6 mutants with single amino acid substitution. In vitro degradation ability of each E6 mutant was equivalent for these tumor suppressors. We investigated whether E6AP is involved in ubiquitin-mediated degradation of hDlg. In vitro binding assay revealed that hDlg formed ternary complex with E6-E6AP complex. The ability of E6 mutants to degrade these tumor suppressors was correlated with their ability to interact with E6AP. Furthermore, hDlg was targeted for in vitro ubiquitination in the presence of both E6 and E6AP. These data revealed that E6AP is extensively involved in the ubiquitin-mediated degradation of E6-dependent substrates as a cellular E3 ubiquitin-protein ligase.

373. Targeting the Central Nervous System by Intravenous Administration of a Targeting Lentiviral Vector

We have developed a targeting lentiviral vector using the modified Sindbis virus envelope protein (Morizono, K et al, Nat. Med. 11: 346|[ndash]|352, Morizono, K et al, J. Virol. 75: 8016|[ndash]|8020). The modified envelope, designated m168, can bind to the Fc region of antibodies, so viral binding and entry can redirected based upon the specificity of the conjugated antibody. We have used this vector to demonstrate targeted transduction of metastatic melanoma in the mouse lung by intravenous administration of the virus. Although the viral vector binds, enters and transduces a wide variety of cells via targeted cell surface molecules, certain cell types are resistant to transduction by the vector in spite of the abundant expression of the targeted molecules. To broaden the application of the vector to more tissues and cell types, we attempted to analyze and overcome the blockage of transduction by the vector. Because the resistant cells are permissive for transduction by VSV-G-pseudotyped lentiviral vectors, the blockage of transduction seems to be present at the step prior to reverse transcription. In addition, the targeting vector efficiently binds to target cells via targeted molecules, so the blockage most likely occurs at fusion between the target cell membrane and the viral envelope. We found that the amount of cholesterol on the target cell membrane controls the efficiency of viral fusion (entry); i.e., depletion of cholesterol from permissive cells decreases the transduction efficiency by the vector, and addition of cholesterol to resistant cells increases the transduction efficiency of the vector. To increase the transduction efficiency to the cells that contain low amounts of cholesterol, we introduced an additional mutation into the E1 gene of the m168 construct, which has been demonstrated to mediate viral fusion in a cholesterol-independent manner. The envelope with the E1 mutation, designated 2.2, dramatically increasedthe transduction efficiency to cholesterol-depleted cells. The 2.2-pseudotyped vector also more efficiently transduces the previously resistant cells in an antibody-dependent manner, including primary peripheral blood mononuclear cells, and human umbilical vein endothelial cells. The 2.2-pseudotyped vector was also tested in an in vivo setting. The virus conjugated with the anti-transferrin receptor was administered intravenously into mice to target blood-brain barrier capillary endothelial cells previously known to express abundant amounts of the transferrin receptor. The 2.2 pseudotype expressed the firefly luciferase transgene at the location of the brain and spine, in an antibody-dependent manner, as demonstrated by cooled coupled charged device imaging. We are trying to define the specific cell types transduced in the central nervous system (CNS) tissues by immunohistochemical analysis, as well as delivery of therapeutic molecules into the CNS.

Lessons from Genetic Disorders of Branched-Chain Amino Acid Metabolism1–3

Genetic disorders of BCAA metabolism produce amino acidopathies and various forms of organic aciduria with severe clinical consequences. A metabolic block in the oxidative decarboxylation of BCAA caused by mutations in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) results in Maple Syrup Urine Disease (MSUD) or branched-chain ketoaciduria. There are presently five known clinical phenotypes for MSUD, i.e., classic, intermediate, intermittent, thiamin-responsive, and dihydrolipoamide dehydrogenase (E3)-deficient, based on severity of the disease, response to thiamin therapy, and the gene locus affected. Reduced glutamate, glutamine, and gamma-aminobutyrate concentrations induced by the accumulation of branched-chain alpha-ketoacids in the brain cortex of affected children and neonatal polled Hereford calves are considered the cause of MSUD encephalopathies. The long-term restriction of BCAA intake in diets and orthotopic liver transplantation have proven effective in controlling plasma BCAA levels and mitigating some of the above neurological manifestations. To date, approximately 100 mutations have been identified in four (branched-chain alpha-ketoacid decarboxylase/dehydrogenasealpha [E1alpha], E1beta, dihydrolipoyl transacylase [E2], and E3) of the six genes that encode the human BCKDC catalytic machine. We have documented a strong correlation between the presence of mutant E2 proteins and the thiamin-responsive MSUD phenotype. We show that the normal E1 component possesses residual decarboxylase activity, which is augmented by the binding to a mutant E2 protein in the presence of the E1 cofactor thiamin diphosphate. Our results provide a biochemical model for the effectiveness of thiamin therapy to thiamin-responsive MSUD patients.

Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli

BackgroundOncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 regions are modified in a time consuming sequential fashion.ResultsBased on the widely used adenoviral cloning system AdEasy we generated a novel transfer vector that allows efficient and rapid generation of conditionally replication-competent adenovirus type 5 based vectors with the viral E1 and E4 genes under the transcriptional control of heterologous promoters. For insertion of the promoters of interest our transfer vector has two unique multiple cloning sites. Additionally, our shuttle plasmid allows encoding of a transgene within the E1A transcription unit. The modifications, including E1 mutations, are introduced into the adenoviral genome by a single homologous recombination step in Escherichia coli. Subsequently infectious viruses are rescued from plasmids. As a proof-of-concept we generated two conditionally replication-competent adenoviruses Ad.Ki•COX and Ad.COX•Ki with the promoters of the Ki-67 protein and the cyclooxygenase-2 (COX-2) driving E1 and E4 and vice versa.ConclusionWe demonstrated with our cloning system efficient generation of double heterologous promoter controlled oncolytic adenoviral vectors by a single homologous recombination step in bacteria. The generated viruses showed preferential replication in tumor cells and in a subcutaneous HT-29 colon cancer xenograft model the viruses demonstrated significant oncolytic activity comparable with dl327.

Regulation of Phosphate Homeostasis by MicroRNA in Arabidopsis

In this study, we reveal a mechanism by which plants regulate inorganic phosphate (Pi) homeostasis to adapt to environmental changes in Pi availability. This mechanism involves the suppression of a ubiquitin-conjugating E2 enzyme by a specific microRNA, miR399. Upon Pi starvation, the miR399 is upregulated and its target gene, a ubiquitin-conjugating E2 enzyme, is downregulated in Arabidopsis thaliana. Accumulation of the E2 transcript is suppressed in transgenic Arabidopsis overexpressing miR399. Transgenic plants accumulated five to six times the normal Pi level in shoots and displayed Pi toxicity symptoms that were phenocopied by a loss-of-function E2 mutant. Pi toxicity was caused by increased Pi uptake and by translocation of Pi from roots to shoots and retention of Pi in the shoots. Moreover, unlike wild-type plants, in which Pi in old leaves was readily retranslocated to other developing young tissues, remobilization of Pi in miR399-overexpressing plants was impaired. These results provide evidence that miRNA controls Pi homeostasis by regulating the expression of a component of the proteolysis machinery in plants.

Papillomavirus E2 Protein Induces Expression of the Matrix Metalloproteinase-9 via the Extracellular Signal-Regulated Kinase/Activator Protein-1 Signaling Pathway

Papillomaviruses are involved in the development of cancers of the female cervix, head and neck, and skin. An excellent model to study papillomavirus-induced tumor induction and progression is the New Zealand White rabbit, where the skin is infected with the cottontail rabbit papillomavirus (CRPV). This leads to the formation of benign tumors that progress into invasive and metastasizing carcinomas without the need for cofactors. We have shown previously that specific mutations in the transactivation domain of the transcription/replication factor E2 cause a dramatic loss in the tumor induction efficiency of the viral genome and a major deficiency in tumor progression as we show now. By comparing wild-type (WT) and mutant E2-induced skin tumors, we found high levels of matrix metalloproteinase-9 (MMP-9) protein and transcripts in WT CRPV-E2-induced tumors in contrast to certain mutant CRPV-E2-induced papillomas and normal uninfected skin. Stable cell lines and reporter assays revealed that E2 from different papillomavirus types is able to transactivate the MMP-9 promoter via the promoter-proximal activator protein-1 (AP-1) site as shown in reporter gene assays with mutant MMP-9 promoter constructs. Furthermore, WT E2 but not mutant E2 strongly transactivated a minimal promoter reporter construct with multiple AP-1 sites. The MMP-9 protein induced in cells expressing E2 degrades collagen matrices as measured in Matrigel-based invasion/mobility assays. E2-induced MMP-9 expression can be blocked by a chemical inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (PD 098059), suggesting that E2 activates the MAPK/ERK signaling pathway, which is further supported by the induction of ERK1 in CRPV-E2-transfected cells.

Induction of tetraploidy through loss of p53 and upregulation of Plk1 by human papillomavirus type-16 E6

Cancer cells are insensitive to many signals that inhibit growth of untransformed cells. Here, we show that primary human epithelial cells expressing human papillomavirus (HPV) type-16 E6/E7 bypass arrest caused by the DNA-damaging drug adriamycin and become tetraploid. To determine the contribution of E6 in the context of E7 to the resistance of arrest and induction of tetraploidy, we used an E6 mutant unable to degrade p53 or RNAi targeting p53 for knockdown. The E6 mutant fails to generate tetraploidy; however, the presence of E7 is sufficient to bypass arrest while the p53 RNAi permits both arrest insensitivity and tetraploidy. We published previously that polo-like kinase 1 (Plk1) is upregulated in E6/E7-expressing cells. We observe here that abnormal expression of Plk1 protein correlates with tetraploidy. Using the p53 binding-defective mutant of E6 and p53 RNAi, we show that p53 represses Plk1, suggesting that loss of p53 results in tetraploidy through upregulation of Plk1. Consistent with this hypothesis, overexpression of Plk1 in cells generates tetraploidy but does not confer resistance to arrest. These results support a model for transformation caused by HPV-16 where bypass of arrest and tetraploidy are separable consequences of p53 loss with Plk1 required only for the latter effect.