Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12, 25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett''s esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36, 40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G₁-like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yields binucleate cells with 4C DNA content, is also a potential mechanism for polyploidy formation (31).The postmitotic checkpoint becomes activated when cells with an intact spindle assembly checkpoint become arrested during mitosis for a prolonged period of time and eventually adapt to the checkpoint, exit mitosis without cleavage, and progress into a G₁-like state with 4C DNA content (19, 22). The cells are prevented from continuing through the cell cycle and replicating their DNA by a proposed p53- and pRb-dependent postmitotic checkpoint (18, 19).High-risk types of HPV (of which HPV-16 is the most prevalent) are commonly associated with lesions that can progress to cervical carcinoma, which is one of the leading causes of cancer death in women worldwide (42). The transforming properties of high-risk HPVs primarily reside in the E6 and E7 oncogenes (reviewed in reference 7). The ability of high-risk HPV E6 and E7 proteins to promote the degradation of p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (6, 30). Expression of the high-risk HPV E6 and E7 oncogenes in human keratinocytes leads to polyploidy, which is enhanced by DNA damage and by activation of the spindle checkpoint through microtubule disruption (15, 27, 37, 38).Previously, it was thought but not directly shown that high-risk E6 and E7 induce polyploidy in response to microtubule disruption by abrogating the spindle checkpoint and that degradation of the tumor suppressor p53 by E6 is the mechanism by which E6 accomplishes this polyploidy formation (27, 37, 38). Others have proposed that E7 may play a role in stimulating DNA rereplication that occurs prior to mitosis initiation and polyploidy formation (20). Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint (21). Instead, E6 abrogates the postmitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy (21). The mechanism by which HPV E7 induces polyploidy remains to be determined. In this study, we investigate these possible mechanisms through which HPV-16 E7 induces polyploidy formation.     

The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.Human papillomaviruses (HPVs) are associated with several human cancers, most notably human cervical cancer, the second most common cancer among women worldwide (43). Papillomaviruses cause proliferative squamous epithelial lesions, and more than 100 HPV types have been described (14). The HPV types associated with mucosal squamous epithelial lesions have been further classified into high- or low-risk types based on the propensity for the lesions with which they are associated to progress to cancer. Among the high-risk HPV types, HPV type 16 (HPV16) and HPV18 account for approximately 70% of cervical cancers (43). The high-risk HPV types carry two genes, the E6 and E7 genes, which have oncogenic properties and are always expressed in HPV-positive cancers. E6 and E7 interfere with the p53 and retinoblastoma (pRB) tumor suppressor pathways, respectively, and contribute directly to cell cycle alterations, protection from apoptosis, and transformation (14). The dysregulated expression of the E6 and E7 oncoproteins is an important step in the progression from a preneoplastic stage to cancer in HPV-infected cells and is often a consequence of the integration of the viral genome into the host chromosome.The interaction between E6 and p53 is mediated by the E3 ubiquitin ligase E6AP (15). E6, p53, and E6AP form a complex in which E6 directs the ligase activity of E6AP to p53, thereby targeting p53 for ubiquitin-mediated degradation (36). E6, however, has a number of other cellular partners and other functions. For instance, the C terminus of the high-risk E6 protein contains a PDZ binding motif (20, 25) that mediates the interaction with several PDZ domain-containing proteins, including discs large (Dlg), Scribble (Scrib), the MAGI family of proteins, MUPP1, and PATJ (9, 10, 29). Some of these proteins are also targeted for degradation in an E6AP-dependent manner (22, 29). While the major mechanism of oncogenesis revolves around E6''s ability to inhibit the proapoptotic effects of p53, recent work involving the PDZ domain proteins indicates that these interactions are also important to the oncogenic potential of E6 (38, 41). Furthermore, E6 has been reported to bind a number of other cellular proteins, including but not limited to Bak, CBP/p300, c-Myc, E6TP1, hADA3, IRF3, MCM7, PTPH1, and TNF-R1 (7, 8, 17, 23, 24, 32, 35, 39, 40). The importance of the binding of several of these proteins with regard to the transformation or other functions of E6 remains to be established. E6 itself is thought to be targeted for degradation by an ubiquitin-proteasome pathway (18), although how E6 protein stability is regulated has not been well studied.Many of the E6 binding partners have been identified using purified bacterially expressed E6 fusion proteins and cell lysates from various cell types or using yeast two-hybrid screenings. While some of these interactions with E6 have been validated, the physiologic relevance of a number of proposed E6 targets remains undetermined. In an effort to identify E6-interacting proteins, perhaps under more physiologic conditions, we employed tandem affinity purification (TAP) using tagged HPV16 E6 stably expressed in the HPV16-positive cervical cancer cell line SiHa. We have discovered several new interacting proteins, including an interaction between E6 and the cellular deubiquitylating enzyme (DUB) USP15. USP15 is not targeted for degradation by E6, but we found that USP15 stabilizes E6 protein levels, suggesting that E6 may itself be a target for USP15 DUB activity.     

Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect keratinocytes of the differentiating epithelium of the skin or mucosa (reviewed in references 4 and 63). Of more than 150 different HPV types identified thus far, about 25 infect the anogenital region (9). The low-risk types, such as HPV11 and HPV6, are associated with the development of genital warts, while the high-risk types, such as HPV16, -18, and -31, cause high-grade lesions that can progress to invasive cervical carcinoma (17, 38, 61).The HPV life cycle is coupled with the differentiation program that keratinocytes undergo in the epithelium. After infection of the basal cell layer of the epithelium, the virus establishes and maintains its genome as an extrachromosomal element (episome) in the nucleus of infected cells. While the viral episome is maintained at low levels in basal cells, its amplification to a high copy number is trigged in the upper layers of the epithelium by the action of the viral oncogenes E6 and E7 and the differentiation of the infected keratinocytes (reviewed in reference 21). Replication of the HPV genome relies on the viral proteins E1 and E2 and the host DNA replication machinery. Viral DNA replication is initiated by the binding of E2 to specific sites on the viral origin where it facilitates the recruitment and assembly of E1 into a double hexamer that is required to unwind DNA ahead of the bidirectional replication fork (3, 14, 15, 31, 33, 36, 43-45, 52, 60). In addition to its helicase activity, E1 interacts with several cellular replication factors, including polymerase α-primase, replication protein A (RPA), and topoisomerase I, to replicate the viral episome (5, 6, 19, 32, 35, 39).E1, which belongs to helicase superfamily III (SF3) (22, 26), can be divided into three functional regions. Its C-terminal domain has ATPase and helicase activity and can self-assemble into hexamers. It is also this domain that is contacted by E2 to recruit E1 at the origin (50, 57, 58). The middle portion of E1 encompasses the origin-binding domain (OBD) that binds and dimerizes on specific sequences in the origin (55, 56). We and others previously found that a fragment of E1 containing only the C-terminal enzymatic domain and the OBD is capable of supporting viral DNA replication in vitro but is inactive in vivo (2, 51). This suggested that the N-terminal region of E1 plays an essential regulatory function in vivo. As such, it has been shown for HPV11 E1 that this region contains a cyclin E/A-Cdk2 (cyclin-dependent kinase 2) binding motif (CBM), a bipartite nuclear localization signal (NLS) and an CRM1-dependent nuclear export signal (NES), which together regulate the nucleocytoplasmic shuttling of the protein (10, 30, 34). Specifically, it has been shown that phosphorylation of HPV11 E1 on three serine residues within its N-terminal region inhibits its nuclear export (10, 62). Interestingly, bovine papillomavirus (BPV) E1 was also shown to shuttle between the nucleus and the cytoplasm in a phosphorylation-dependent manner. In this case, however, Cdk2 phosphorylation was found to promote, rather than inhibit, the export of the viral helicase (24). This apparent discrepancy between HPV11 and BPV E1 prompted us to examine the regulation of a third E1 protein, specifically that of the high-risk HPV31.We report here that HPV31 E1 also shuttles between the nucleus and the cytoplasm through its conserved NLS and NES. We determined that nuclear export of HPV31 E1 is dependent on the CRM1 export pathway and is inhibited by Cdk2 phosphorylation of serines 92 and 106. We also found that nuclear export of E1 is not required for transient viral DNA replication and thus investigated its role in viral genome maintenance and amplification in immortalized keratinocytes. In contrast to the wild type (WT), a mutant genome carrying a defective E1 NES was poorly maintained and progressively lost upon cell division, indicating that nuclear export of E1 is required for long-term maintenance of the viral episome. Because nuclear export of E1 is not required for viral DNA replication per se but needed for episomal maintenance over several cell divisions, we investigated the possibility that continuous accumulation of E1 into the nucleus is detrimental to cellular proliferation. In support of this possibility, we found that the accumulation of E1 at high levels in the nucleus impedes cellular proliferation by delaying cell cycle progression in the S phase. In addition, we found that this delay was alleviated when nuclear export of E1 was increased. Altogether, these results suggest that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.     

The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A₁ (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.High-risk human papillomaviruses (HPVs) are the causative agent of cervical cancer (63) and HPV type 16 (HPV-16) is associated with a majority of cervical malignancies worldwide (13). HPV-16 encodes three oncoproteins: E5, E6, and E7. While the contributions of E6 and E7 to cellular immortalization and transformation have been characterized in detail (20), the role of HPV-16 E5 (16E5) is poorly understood (53). Nevertheless, a number of studies suggest that 16E5 does contribute to the development of cervical cancer. Most high-risk HPV types encode an E5 protein (48), and targeted expression of the three HPV-16 oncogenes in basal epithelial cells of transgenic mice (4) leads to a higher incidence of cervical cancer than does the expression of E6 and E7 alone (44). In addition, targeted epithelial expression of 16E5 (without E6 and E7) in transgenic mice induces skin tumors (21). It may be noteworthy that unlike high-risk HPV-18, which integrates into the host DNA and potentially disrupts E5 gene expression (20, 64), the HPV-16 genome often persists in episomal form in malignant lesions (12, 16, 24, 36, 42).Biological activities of 16E5 that may facilitate carcinogenesis include evading host immune detection by interfering with the transport of antigen-presenting major histocompatibility complex (MHC) class I molecules to the cell surface (6), promoting anchorage-independent growth (33, 41, 52) and disrupting gap junctions responsible for cell-cell communication (37, 58). The 16E5 phenotype most frequently linked to the development of cancer is enhanced ligand-dependent activation of the epidermal growth factor receptor (EGFR) (15, 41, 46, 52). 16E5 stimulates EGF-dependent cell proliferation in vitro (7, 33, 40, 41, 52, 60) and in vivo (21), which might expand the population of basal or stemlike keratinocytes and thereby increase the probability that some of these cells would undergo malignant transformation. A number of studies indicate that 16E5 may enhance ligand-dependent EGFR activation by interfering with the acidification of early endosomes containing EGF bound to activated EGFRs (17, 51, 57). It has been hypothesized that 16E5 inhibits the H⁺ V-ATPase responsible for maintaining an acidic luminal pH in late endosomes and lysosomes (28) by associating with the V-ATPase 16-kDa “c” subunit (16K) (1, 5, 14, 22, 46) and disrupting assembly of the V-ATPase integral (V_o) and peripheral (V_i) subcomplexes (10). In contrast, Thomsen et al. (57) reported that 16E5 inhibits early endosome trafficking in fibroblasts by completely depolymerizing actin microfilaments.Due to the unavailability of antibodies that recognize native 16E5 and 16K, direct association of 16E5 with 16K has only been observed by overexpressing epitope-tagged forms of both proteins in vitro (5, 46) or in vivo (1, 14, 22). It is uncertain, therefore, whether these associations occur when the proteins are expressed at “physiological” levels. In yeast, both wild-type 16E5 (10) and several 16E5 mutants that associate with 16K in COS cells (1) inhibit vacuolar acidification, although another study in yeast concludes the opposite (5). 16K is a component of the V-ATPase V_o subcomplex, which is assembled in the endoplasmic reticulum (ER) (28), and 16E5 localizes to the ER and nuclear envelope in epithelial cells (32, 54). Thus, the export of V_o from the ER could potentially be inhibited by a significant level of 16K binding to 16E5, although the differential alkalinization of endosomes rather than the Golgi apparatus (17) would require specificity for those proton pumps directed to those sites.In the present study, we generated an antibody against native 16K and used it to determine whether 16K/16E5 complexes formed in primary keratinocytes. We also synthesized a new pH-sensitive fluorescent EGF conjugate to evaluate whether there was a correlation between E5-induced EGFR activation, trafficking and endosome alkalinization. Finally, we simultaneously monitored EGFR endocytic trafficking (using pH-insensitive fluorescent EGF), endosome fusion (using fluorescent EGF and dextran), and the status of cellular filaments and microtubules to evaluate whether E5 might disrupt some of these structures that mediate vesicle transport.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14, 15, 30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14, 15, 30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ∼8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a T=7 icosahedral lattice (2, 9, 17, 19, 36, 42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6, 12, 25, 40, 43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7, 20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation and infectivity of HPV-infected tissues are redox dependent in that they can be manipulated via the treatment of organotypic tissues with oxidized glutathione (GSSG), which is concentration and temporally dependent.     

The Canine Papillomavirus E5 Protein Signals from the Endoplasmic Reticulum

The recently discovered Canis familiaris papillomavirus (PV) type 2 (CfPV2) provides a unique opportunity to study PV gene functions in vitro and in vivo. Unlike the previously characterized canine oral PV, CfPV2 contains an E5 open reading frame and is associated with progression to squamous cell carcinoma. In the current study, we have expressed and characterized the CfPV2-encoded E5 protein, a small, hydrophobic, 41-amino-acid polypeptide. We demonstrate that, similar to the E5 protein from high-risk human PV type 16, the CfPV2 E5 protein is localized in the endoplasmic reticulum (ER) and that its expression decreases keratinocyte proliferation and cell life span. E5 expression also increases the percentage of cells in the G₁ phase of the cell cycle, with a concomitant decrease in the percentage of cells in S phase. To identify a potential mechanism for E5-mediated growth inhibition from the ER, we developed a real-time PCR method to quantify the splicing of XBP1 mRNA as a measure of ER stress. We found that the CfPV2 E5 protein induced ER stress and that this, as well as the observed growth inhibition, is tempered significantly by coexpression of the CfPV2 E6 and E7 genes. It is possible that the spatial/temporal regulation of E6/E7 gene expression during keratinocyte differentiation might therefore modulate E5 activity and ER stress.Papillomaviruses (PVs) are a large group of DNA tumor viruses that infect differentiated cutaneous and mucosal epithelia in a wide variety of mammalian species. There are nearly 200 types of human PVs (HPVs) (61), some of which are termed high risk (e.g., HPV type 16 [HPV-16]) and have the potential to immortalize primary cells and facilitate malignant progression to cervical cancer (52). An estimated 20 million cases of HPV infection occur each year in the United States alone, and cervical cancer is the second most common cause of cancer deaths among women worldwide. In general, PV infections are species specific, making it impossible to study the in vivo life cycle of HPV and the roles of its encoded proteins in viral replication and tumorigenesis. However, a few animal models do exist and the canine oral PV (COPV) has been helpful in mimicking certain biological properties of the high-risk mucosatropic HPVs, leading to the development of highly effective prophylactic vaccines (39, 49, 56). Although COPV mimics the mucosal tropism of the high-risk HPVs, it rarely progresses to cancer and lacks one of the early viral genes that may play an important role in tumorigenesis, E5. Recently, a new canine PV (Canis familiaris PV type 2 [CfPV2]) was isolated from the footpads of dogs (43). Unlike COPV, CfPV2 induces epidermal tumors and, when persistent, these benign infections progress to squamous cell carcinoma and metastasize widely. CfPV2 also encodes an E5 protein. In general, PV E5 proteins are small hydrophobic oncoproteins that localize to the endoplasmic reticulum (ER) or Golgi membranes (11, 16) but have limited amino acid sequence homology. Numerous cellular binding partners have been described for HPV-16 E5 proteins, including the V-ATPase 16-kDa subunit (1, 16), the nuclear import protein karyopherin beta 3 (25), the ER-resident protein Bap31 (40), proteins involved in zinc transport (ZnT1, EVER1, and EVER2) (27, 35), erbB4 (24), and HLA I (2). The HPV-16 E5 protein alters signaling pathways, predominantly the epidermal growth factor receptor (EGFR) pathway (17, 21, 46, 58); induces koilocytosis in cooperation with the E6 protein (26); and alters the plasma membrane expression of caveolin (47), HLA (3), and ganglioside GM1 (47). The last two changes might explain the ability of HPV-16-infected cells to circumvent detection by the host immune response and initiate tumor formation (3, 4, 21, 36, 46, 47).To provide a foundation for future in vivo studies, we initiated a series of in vitro experiments to define the intracellular localization and biological activity of CfPV2 E5. The current study demonstrates that CfPV2 E5 exhibits several properties of the HPV-16 E5 protein, including ER localization and inhibition of cell proliferation. A novel finding is that CfPV2 E5 activates the ER stress-signaling pathway, which may explain some of E5''s growth-related activities.