The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.During the past decade protein degradation has become increasingly recognized as a critical mechanism by which cells regulate a number of fundamental processes (reviewed in references 37, 57, and 59). Degradation frequently involves one of a variety of E3 ubiquitin ligase complexes in which a substrate recognition component introduces the target protein for ubiquitination and subsequent degradation by proteasomes (reviewed in reference 59). Several types of these complexes involve a member of the cullin family (reviewed in reference 59), and a considerable amount of information is known about those containing Cul2 or Cul5. In these cases the substrate recognition module is linked via elongins B and C to a subcomplex containing Cul2 or Cul5 and the RING protein Rbx1 (34, 58). This complex interacts with an E2 conjugating enzyme, often either Cdc34 or Ubc5, to conjugate ubiquitin chains to the substrate (44). With both Cul2- and Cul5-based complexes interaction with elongins B and C occurs via a single BC box sequence (42). The presence of either Cul2 or Cul5 is generally determined through the presence in the substrate recognition protein of specific Cul2- or Cul5-box sequences (35).Many viruses have evolved to encode products that inhibit cellular E3 ligases to protect important viral or cellular species or, in some cases, that highjack these cellular complexes to target key substrates for degradation, including components of cellular host defenses, to facilitate the infectious cycle (reviewed in reference 4). These strategies are quite common among the small DNA tumor viruses (7), and one of the most studied examples is the complex formed by the human adenovirus E4orf6 and E1B55K proteins. These proteins have been known for some time to interact (69) and to reduce the levels of the p53 tumor suppressor in infected cells (14, 47, 48, 62, 72, 73). In addition, they were shown to function in concert to block nuclear export of cellular mRNAs late in infection (2, 6, 29, 60) and to enhance the selective export of late viral mRNAs (2, 26, 29, 60, 78). Our group showed that the human adenovirus serotype 5 (Ad5) E4orf6 product interacts with several proteins (13), including components of what was at the time a unique Cul5-based E3 ubiquitin ligase containing elongins B and C and Rbx1 that degrades p53 (61). Curiously, Ad5 E4orf6 contains three BC boxes that we believe make it highly efficient in highjacking cellular elongin B/C complexes (8, 17, 41). The mechanism of selective recruitment of Cul5 by the Ad5 complex remains unknown as E4orf6 lacks a Cul5-box (17, 41). E1B55K seems to function as the substrate recognition module and, of considerable interest, both its association with E4orf6 and induction of selective late viral mRNA transport was found to depend on formation of the E3 ubiquitin ligase complex, suggesting that additional degradation substrates must exist (8, 9). This idea is not surprising since viruses, especially the small DNA tumor viruses, often evolve gene products that target multiple critical cellular pathways (32). In fact two additional E1B55K-binding substrates have now been identified, Mre11 from the MRN DNA repair complex (8, 75), and DNA ligase IV (3), the degradation of which prevent formation of viral genome concatemers, thus enhancing packaging of progeny DNA. Degradation of p53 has been suggested to promote enhanced progeny virus production by preventing the early apoptotic death of infected cells due to the stabilization of p53 by the viral E1A products (reviewed in reference 66). Nevertheless, degradation of these substrates seems unlikely to explain the observed effects on mRNA transport, suggesting that still more substrates remain to be identified. Although the studies described in the present report were in part launched to identify such substrates, as will become clear below, these targets remain to be identified.In an attempt to identify new substrates of the Ad5 E4orf6/E1B55K E3 ubiquitin ligase complex, a proteomics-based approach was initiated involving two-dimensional difference gel electrophoresis (2D-DIGE) analysis and subsequent mass spectrometry. As is well known, this technique has the advantage of improved sensitivity and accuracy provided by its ability to separate samples under two different conditions on a single gel together with a reference sample, thus reducing significantly the analytical coefficient of variation. It allows the quantification of differentially abundant proteins in complex biological samples, providing a tool to detect decreases in the levels of proteins in the cell due to targeted proteolytic degradation. We report here our attempts to identify substrates of the Ad5 E4orf6/E1B55K complex by comparing the proteomes of human non-small cell lung carcinoma H1299 cells expressing, by means of adenovirus vectors, both E1B55K and E4orf6 proteins or E4orf6 protein alone. Ten candidate proteins were identified, most having functions seemingly unrelated to our current understanding of the roles of the E4orf6/E1B55K complex. At least three showed promising features characteristic of substrates, and one has now been confirmed in a parallel study to be a bone fide E4orf6/E1B55K substrate (20). We suggest that this approach could be utilized to identify candidate substrates, among relatively high abundance proteins, that are degraded by other viral cullin-based E3 ubiquitin ligase complexes.     

Adeno-Associated Virus Small Rep Proteins Are Modified with at Least Two Types of Polyubiquitination

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.Together, adenovirus type 5 (Ad5) early gene products E1a, E1b-55k, E2a, E4orf6, and virus-associated (VA) RNA can support efficient replication of adeno-associated virus (AAV) (4, 31). E4orf6 and E1b-55k are known to interact with cellular cullin 5 (cul5), elongins B and C, and the ring box protein Rbx1 to form an E3 ubiquitin ligase complex that specifically targets a small population of cellular proteins for degradation by the proteasome (1, 7, 21, 22, 24, 27). This property has been implicated in a number of functions presumed to be required for both Ad and AAV replication (3, 8-10, 17, 23, 24, 34, 35).Previously, only p53, Mre11, DNA ligase IV, and integrin α3 had been shown to be substrates of the Ad5 E3 ubiquitin ligase complex (1, 7, 21, 22, 24, 27); however, we have recently shown (16, 17) that the small Rep proteins and capsid proteins of AAV5 are also degraded in the presence of Ad E4orf6 and E1b-55k in a proteasome-dependent manner. These proteins were restored to levels required during infection by the action of VA RNA (17). The targeting for degradation of AAV5 protein by the E4orf6/E1b-55k E3 ubiquitin ligase complex required functional BC-box motifs in E4orf6 and could be inhibited by depletion of the scaffolding protein cullin 5 using directed small interfering RNA (siRNA) (16). In addition, the degradation of AAV5 protein was partially prevented by overexpression of pUBR7, a plasmid that generates a dominant-negative ubiquitin (16). The role this targeted degradation plays in the life cycle of AAV has not yet been clarified; however, E4orf6 mutants that cannot function in this regard do not support AAV replication as well as wild-type E4orf6 (R. Nayak and D. J. Pintel, unpublished data). Degradation of Mre11 by the Ad5 E3 ligase has also been implicated in allowing efficient Ad5 and AAV replication (24). Ubiquitination of AAV Rep proteins during viral infection, however, has not previously been reported.     

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.     

Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein

The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.Human papillomaviruses (HPVs) are small, nonenveloped, double-stranded DNA viruses (25) that are the causative agents of benign and malignant tumors in humans (43). Most cancers of the cervix, vagina, and anus are caused by HPVs, as are a fraction of oropharyngeal cancers (29, 44). HPV type 16 (HPV-16) is the type most frequently found in anogenital cancers (15, 29), including cervical cancer, the most common cancer of women worldwide (44).Some of the biological activities of the HPV-16 E5 protein (16E5) include the augmentation of epidermal growth factor (EGF) signaling pathways (8), stimulation of anchorage-independent growth (38), alkalinization of endosomal pH (11), and alteration of membrane lipid composition (39). 16E5 also exhibits weak transforming activity in vitro (12), induces epithelial tumors in transgenic mice (13), and plays an important role in koilocytosis (20). There are multiple documented intracellular binding targets for 16E5 such as the 16-kDa subunit of the vacuolar H⁺-ATPase (7, 36), the heavy chain of HLA type I (1), EGF receptor family member ErbB4 (6), calnexin (16), the zinc transporter ZnT-1 (21), the EVER1 and EVER2 transmembrane channel-like proteins that modulate zinc homeostasis (21, 31), the nuclear import receptor family member karyopherin β3 (KNβ3) (19), and BAP31, which was previously reported to contribute to B-cell receptor activation (35).16E5 is a small, hydrophobic protein that localizes to intracellular membranes. When overexpressed in COS cells, it is present in the endoplasmic reticulum (ER) and, to a lesser extent, in the Golgi apparatus (7). At a lower level of expression in human foreskin keratinocytes and human ectocervical cells (HECs), 16E5 is present predominantly in the ER (10, 39). 16E5 contains three hydrophobic regions (14, 16, 22, 30, 41), and it was reported previously that the first hydrophobic region determines various biological properties of the protein (16, 22). It was also shown previously that the 16E5 C terminus plays a role in binding to karyopherin β3 (19) and in the formation of koilocytes (20). While theoretical predictions have been made for the topology of E5 in membranes (16), no experimental data exist. However, a recent study suggested that some highly expressed 16E5 localizes to the plasma membrane, with its C terminus exposed externally (18).The aim of the present study was to establish the orientation of 16E5 in the ER membrane. By using immunofluorescence microscopy coupled with differential membrane permeabilization (24, 34), we demonstrate the membrane orientation of an N- and C-terminally tagged, biologically active 16E5 protein. Our results indicate that the N terminus is intralumenal and that the C terminus is cytoplasmic, consistent with a model of E5 being a three-pass transmembrane protein and with current data on the interaction of its C terminus with cytoplasmic proteins.     

PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development

One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.Polypeptide growth factors, such as insulin, insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang1), exert biological functions through binding to their transmembrane receptors that belong to a large family of receptor tyrosine kinases (RTKs) (4). Consequently, the receptor molecules form homo- or heterodimers, and the intracellular tyrosines at the carboxyl termini of the receptors become phosphorylated (37). Numerous distinct adaptor/regulatory proteins, through their Src homologous 2 (SH2) domains, bind to the phosphotyrosines and transduce the signal to downstream pathways, among which are two essential and well-characterized signaling cascades—the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt signaling pathways (4, 13, 37).The regulatory subunit of PI3K, p85, possesses the SH2 domain and can, therefore, bind to phosphotyrosines on the RTKs and subsequently render activation of the catalytic subunit of PI3K, p110 (7, 8). Active p110 phosphorylates phosphoinositide biphosphate (PIP2), turning it into PIP3 that recruits PDK1 and Akt to the cellular membrane, where Akt is phosphorylated at threonine 308 (T308 for Akt1) by PDK (5, 23, 30). The serine 473 (S473) of Akt (Akt1) is phosphorylated by mTOR complex 2 (mTORC2) and other kinases (17, 36). Phosphorylation of Akt at these two amino acids brings it to full activation. In PDK1-deficient embryonic stem (ES) cells, T308 phosphorylation was abolished and most of the Akt activity was lost, although the S473 phosphorylation was intact (40).Akt plays an important role in multiple biological processes, such as cell survival, growth, glucose metabolism, and angiogenesis (2, 12, 14-16, 22, 23, 39, 41-43). In mammals, there are three Akt isoforms, termed Akt 1, -2, and -3. Previously, we generated Akt1- and Akt3-deficient mice and studied their roles in mouse development (2, 15, 39, 42, 43). We found that the Akt1 and -3 double knockout (KO) (DKO) mice were embryonically lethal at around embryonic day 12 (E12) and manifested developmental defects in multiple tissues, including the cardiovascular system (14, 15, 43). These studies suggest that the Akt signaling pathway is involved in cardiovascular development.Other than Akt isoforms, PDK1 also activates another group of AGC family kinases, such as p70 ribosomal S6 kinase (S6K) (32), serum, and glucocorticoid-induced protein kinase (SGK) (26), p90 ribosomal S6 kinase (RSK) (21), and atypical isoforms of protein kinase C (PKC) (31). Comprehensive and intensive mouse genetic studies performed mainly by Alessi and coworkers have confirmed the regulation of these AGC kinases by PDK1 (3, 9, 10, 27-29, 40).PDK1 knockout mice were severely growth retarded and died at around E9.0, indicating an essential role of PDK1 in development (27). However, its function and downstream targets in cardiovascular development are still elusive. To study this, we deleted PDK1 specifically in endothelial cells through Cre recombinase-mediated excision (25). The results have revealed an essential role of PDK1 in vascular remodeling and integrity and in cardiac development through activation of Akt and its downstream target of Snail.     

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.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

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.