The Human Adenovirus E4-ORF1 Protein Subverts Discs Large 1 to Mediate Membrane Recruitment and Dysregulation of Phosphatidylinositol 3-Kinase

Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.


Introduction
Human adenovirus type 9 (Ad9) is a member of the subgroup D adenoviruses that cause eye infections in people [1]. In addition, infection of experimental animals with Ad9 generates estrogendependent mammary tumors, and the E4-ORF1 gene is the primary viral oncogenic determinant [2][3][4]. This viral gene likely evolved from a cellular dUTPase gene, which codes for an enzyme of nucleotide metabolism, and E4-ORF1 and dUTPase share a similar protein fold [5,6]. However, the E4-ORF1 protein lacks dUTPase catalytic activity, indicating functional divergence from dUTPase. Instead, E4-ORF1 functions to activate cellular class IA phosphatidylinositol 3-kinase (PI3K) at the plasma membrane of Ad9-infected human epithelial cells and Ad9-induced experimental tumor cells [7]. This function is conserved in other human adenovirus E4-ORF1 proteins and is essential for Ad9-induced oncogenesis [7]. E4-ORF1 activation of PI3K also enhances productive replication of human adenovirus type 5 (Ad5) by overriding protein translation checkpoints [8,9], prolongs survival of Ad5 vector-infected primary human endothelial cells [10], and modulates lipid and glucose metabolism in human adenovirus type 36-infected cells [11].
Class IA PI3K is a lipid kinase that under normal physiological conditions functions as a key downstream effector of membrane receptors and ras [12]. PI3K exists as a heterodimer composed of p85 regulatory and p110 catalytic subunits. In the cytoplasm, the regulatory subunit stabilizes the catalytic subunit and inhibits its lipid kinase activity. Activated membrane receptors and ras can bind and recruit cytoplasmic PI3K to the plasma membrane, bringing it into contact with the lipid substrate phosphatidylinositol-4,5-bisphosphate (PIP2) and also relieving enzymatic inhibition by the p85 regulatory subunit. PI3K converts PIP2 to the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn recruits PI3K effector proteins Akt and PDK1 to the plasma membrane. At this site, Akt is activated by phosphorylation on threonine 308 (T308) by PDK1 and on serine 473 (S473) by mTORC2. Numerous downstream effectors of Akt act to regulate a broad range of cellular processes that include metabolism, protein synthesis, growth, survival, migration, and proliferation. Notably, the PI3K signaling pathway is one of the most frequently dysregulated pathways in human cancers [13], and PI3K and its downstream effectors are subverted by many pathogenic human viruses to enhance virus-cell and virus-host interactions, such as viral entry, replication, reactivation from latency, and pathogenesis [14]. These observations underscore a prominent role for the PI3K pathway in human disease.
Mutational analyses of the 125-residue Ad9 E4-ORF1 polypeptide identified multiple amino acid residues required for activation of PI3K, promotion of oncogenic transformation in cultured cells, and tumorigenesis in experimental animals [7,[15][16][17]. Some of these crucial residues cluster at the E4-ORF1 carboxyl-terminus [18], which defines an element that mediates direct binding to cellular PSD95/Dlg/ZO-1 (PDZ) domain proteins [19], thereby revealing the first known virus-encoded PDZ domain-binding motif (PBM). The identification of the E4-ORF1 PBM led to the discovery that the E6 and Tax proteins encoded by high-risk HPV or HTLV-1, respectively, also possess a carboxyl-terminal PBM that is crucial for their oncogenic potential [19][20][21][22][23][24]. It is now recognized that many viruses code for PBMcontaining proteins that target a wide variety of cellular PDZ proteins [23].
In cells, E4-ORF1 exists as both a monomer and homo-trimer, each of which targets a different subset of PDZ proteins with distinct functional outcomes [5]. The E4-ORF1 monomer specifically binds and sequesters MUPP1, PATJ, MAGI-1, and ZO-2 within cytoplasmic punctae [25,26,28] and, as a result, disrupts the TJ and causes a loss of cell polarity [27]. Such defects are hallmarks of cancer cells and may directly contribute to carcinogenesis by deregulating normal cellular proliferation and differentiation programs [29][30][31]. More relevant to the current study, the E4-ORF1 trimer specifically binds to Dlg1 [5], which consists of three PDZ domains along with Lin-2 and Lin-7 (L27), Src homology 3 (SH3), and guanylate kinase-homology (GuK) domains, as well as different insertion elements (I1-I5) generated by alternative splicing [32]. While evidence indicates that Dlg1 has a tumor suppressor function [33], Dlg1 +/+ but not mutant Dlg1 2/2 mouse embryo fibroblasts are able to support oncogenic PI3K activation by Ad9 E4-ORF1 [34]. This finding not only revealed an absolute dependence on Dlg1 for this activity but also exposed a previously unrecognized Dlg1 oncogenic function, which may be widely important given that high-risk HPV E6 proteins require Dlg1 to promote invasive properties in cervical carcinoma cells [33,35]. The Dlg1 oncogenic function hijacked by E4-ORF1 derives from a specific Dlg1 splice isoform, Dlg1-I3, which has an I3 insertion element that localizes Dlg1 to the plasma membrane by binding to membrane-associated protein 4.1 [36,37]. Moreover, Dlg1-I3 contributes to E4-ORF1-mediated PI3K activation, at least in part, by recruiting E4-ORF1 to the plasma membrane [34], implying that the Dlg1:E4-ORF1 complex activates PI3K at this site. However, the molecular mechanism of activation has not been determined and remains an important gap in knowledge.
The present study was undertaken to test the hypothesis that PI3K activation depends on an additional undetermined cellular factor recruited by E4-ORF1 into the Dlg1:E4-ORF1 complex. This idea prompted a search for new E4-ORF1-interacting proteins and led to identification of PI3K itself. Our findings revealed that E4-ORF1 binds directly to PI3K and recruits it into the Dlg1:E4-ORF1 complex, thereby forming a Dlg1:E4-ORF1:-PI3K ternary complex that localizes to the plasma membrane and stimulates PI3K signaling. This novel mechanism of PI3K activation may serve as a paradigm to understand how other pathogenic human viruses dysregulate PI3K and how the common viral target Dlg1, and possibly other PDZ proteins, contributes to viral infections and diseases.

Cellular PI3K is a direct target for the adenovirus E4-ORF1 protein
To identify the postulated cellular factor that binds to E4-ORF1 in the Dlg1:E4-ORF1 complex, we subjected HeLa cell lysates to an in vitro pulldown assay with glutathione S-transferase (GST) fused to Ad9 E4-ORF1, followed by a mass spectrometry analysis of associated proteins. Table S1 lists 10 selected identified proteins, eight of which were either identical (Abl1, ELAV1, IGF2BP1, IGF2BP3) or related to (UPF2, LARP1, LARP2, DDX17) Ad5 E4-ORF1-binding proteins reported in a recent proteomic study of DNA tumor virus oncoproteins [38], suggesting that these eight proteins may be genuine E4-ORF1 cellular targets. More importantly, the other two proteins in the list were novel, and their identities as the PI3K regulatory subunits p85a and p85b prompted additional experiments to confirm and extend the finding. We first used immunoblots to examine proteins recovered from a similar pulldown conducted with lysates of human MCF10A cells, an immortalized but non-transformed human mammary epithelial cell line retaining properties of normal breast epithelial cells [39]. The results showed that GST-E4-ORF1 but not GST interacts with p85b and the PI3K catalytic subunit p110a and that neither GST protein associates with the cellular PDZ protein scribble ( Figure 1A). Moreover, GST-E4-ORF1 but not GST also interacted with the purified,

Author Summary
Adenoviruses cause acute illnesses in people, and are additionally utilized both as vehicles to cure genetic diseases, fight cancer, and deliver vaccines, and as tools to discover how cancers develop due to a capacity to generate tumors in experimental animals. The adenovirus E4-ORF1 protein reprograms cell metabolism to enhance virus production in infected cells and promotes cell survival and tumors by activating the important cellular protein phosphatidylinositol 3-kinase (PI3K). How E4-ORF1 activates PI3K is not known, though this function depends on E4-ORF1 binding to the membrane-associated cellular protein Discs Large 1 (Dlg1), which many different viruses evolved to target. In this study, we identify PI3K as a new direct target of E4-ORF1. Results further show that E4-ORF1 binds to PI3K in the cytoplasm and delivers it to Dlg1 at the membrane where the three proteins form a complex that activates PI3K and induces oncogenic growth in cells. This novel molecular mechanism in which adenovirus subverts Dlg1 to dysregulate PI3K may serve as a paradigm to understand PI3K activation mediated by other important pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 in infected cells.
catalytically-active recombinant human p85a:p110a heterodimer ( Figures 1B and 1C). The low binding efficiency of GST-E4-ORF1 seen in these assays is a general property attributed to aggregation of this bacterially expressed fusion protein. These collective data indicated specific and direct binding of Ad9 E4-ORF1 to functional class IA human PI3K.
E4-ORF1 mediates Dlg1-dependent PI3K activation and protein elevation in human epithelial cells To investigate whether E4-ORF1 associates with endogenous PI3K in human epithelial cells, we generated and characterized MCF10A lines transduced either by the empty pBABE retroviral expression vector (vector cells) or by pBABE encoding the wild-type (wt) Ad9 E4-ORF1 protein (wtORF1 cells), the Ad9 E4-ORF1 PBM mutant V125A protein unable to bind PDZ proteins (V125A cells), or the mutant rasV12 protein that lacks a PBM but activates PI3K and the Raf/MAP kinase pathway (rasV12 cells) [7,19].
Consistent with previous results in rodent fibroblast lines [7], immunoblots of extracts from the cell lines revealed higher levels of activated PI3K effector Akt dually phosphorylated at T308 and S473 (P-Akt) in wtORF1 and rasV12 cells than in vector and V125A cells (  [34]. Compared to vector and V125A cells, wtORF1 and rasV12 cells also displayed higher levels of p85a, p85b, and p110a ( Figure 2, compare lanes 1-2 to lanes 3 and 5). Moreover, treatment of wtORF1 and rasV12 cells with the PI3K inhibitor drug LY294002 (LY) returned the high P-Akt levels to those of vector cells, yet had little or no effect on the high p85 and p110 levels ( Figure 2, compare lane 1 to lanes 3-4 and 5-6). In contrast, stable expression of a short hairpin RNA (shRNA) that depleted Dlg1 decreased the high levels of P-Akt, p85, and p110 in wtORF1 cells but not in rasV12 cells (compare Figure 3A, lanes 3-4 to Figure 3B, lanes 3-4). From cumulative immunoblots, we quantified pertinent protein level differences for wtORF1 or rasV12 cells versus vector cells, as well as for wtORF1 cells transduced with the Dlg1 shRNA vector versus the scrambled shRNA vector (Tables S2, S3, and S4). Taken together, the data showed that while E4-ORF1 and rasV12 similarly elevate p85a, p85b, and p110a levels and activate Akt in a PI3Kdependent manner in human epithelial cells, E4-ORF1 differs from rasV12 in its dependence on both a PBM and Dlg1 for these activities and in not activating the Raf/MAP kinase pathway.
We previously reported that ras is required for E4-ORF1induced PI3K activation in mouse fibroblasts [34]. To test whether this finding would extend to the current system, we transfected MCF10A cells with a wt E4-ORF1 expression plasmid alone or in combination with an expression plasmid encoding dominant-negative mutant rasN17. The data showed that rasN17 over-expression blocked PI3K activation induced by mutant rasv12 ( Figure S2A, compare lanes 3-4) but not E4-ORF1 ( Figure S2B, compare lanes 3-4). These findings suggested that ras does not mediate E4-ORF1-induced PI3K activation in MCF10A cells.
In a reciprocal experiment, we IPed Dlg1 from lysates of the same cell lines and tested for coIP of E4-ORF1, p110a, p85a, and p85b ( Figure 5). Here we observed coIP of the wt E4-ORF1 protein but not PBM mutant V125A or T123D protein with Dlg1 as expected, and also co-IP of p110a, p85a, and p85b with Dlg1, but again only from wtORF1 cells (compare lane 10 to lanes [11][12]. Significantly, the latter results with vector, wtORF1, and V125A cells were mirrored when Dlg1 was IPed from lysates of mock-infected MCF10A cells or MCF10A cells infected with wt Ad9 virus or mutant Ad9 virus having the PBM mutation V125A introduced into the E4-ORF1 gene (Ad9-V125A) ( Figure 6A, compare lane 5 to lanes 4 and 6). As controls, we verified that the infection with each virus was comparable by examining E4-ORF1 protein levels ( Figure 6A), viral major capsid protein hexon accumulation ( Figure 6B), and cytopathic effects ( Figure 6C). The comparable hexon protein accumulation was consistent with our failure to detect a replication defect for the Ad9-V125A Depletion of Dlg1 has no effect on either PI3K activation or PI3K protein elevation in rasV12 cells. Extracts of rasV12 cells transduced with pSUPER-Dlg1 shRNA (+) or pSUPER-Dlg1 scrambled control shRNA (2) were analyzed in an immunoblot assay with antibodies to the indicated proteins. doi:10.1371/journal.ppat.1004102.g003 mutant virus in MCF10A cells (unpublished results). In addition, wt Ad9 virus-infected cells showed higher levels of activated P-Akt than either Ad9-V125A virus-or mock-infected cells ( Figure 6A; Tables S7 and S8), similar to previous results examining P-Akt activation in human A549 cells mock-infected or infected with wt Ad9 or mutant Ad9-IIIA virus, which like the Ad9-V125A virus encodes an E4-ORF1 gene with a disrupted PBM [7]. We also observed decreased Dlg1 levels in cells infected with either wt Ad9 virus or Ad9-V125A virus compared to mock-infected cells ( Figure 6A, compare lane 1 and lanes 2-3). A similar effect was detected in rasV12-expressing cells ( Figure 3B, compare lanes 1 and 3), whereas the modest reduction in Dlg1 levels seen in wtORF1 cells compared to vector cells in Figure 3A was not reproducible. We do not yet understand how Dlg1 is downregulated in adenovirus-infected cells and RasV12-expressing cells.
The biochemical data presented above importantly showed that in human epithelial cells, either stably expressing wt E4-ORF1 protein or lytically infected with wt Ad9 virus, the E4-ORF1 protein binds the PI3K p85:p110 heterodimer in a PBM-independent manner and, in conjunction with separate PBMdependent binding to Dlg1, tethers these two cellular factors together to form a Dlg1:E4-ORF1:PI3K ternary complex. Moreover, formation of this ternary complex strictly correlated with E4-ORF1-mediated PI3K/Akt activation as we observed the complex in wtORF1 cells and wt Ad9 virus-infected cells but not in V125A cells, T123D cells, or mutant Ad9-V125A virus-infected cells.
Nonetheless, immunoblots of lysates from wt Ad9 virus-and mutant Ad9-V125A virus-infected cells revealed some differences from corresponding wtORF1 and V125A cells. Whereas the levels of p85a, p85b, and p110a were higher in wtORF1 cells than in V125A and vector cells (Figure 2), these protein levels were higher in both wt Ad9 virus-and Ad9-V125 virus-infected cells than in mock-infected cells, with comparable p85a and p110a elevation in the virus-infected cells and somewhat higher p85b elevation in wt Ad9 virus-infected cells than in Ad9-V125 virusinfected cells ( Figure 6A; Tables S7 and S8). These results suggested that, in adenovirus-infected cells, p85 and p110 are upregulated by two distinct viral functions, E4-ORF1 and an  . E4-ORF1 binds endogenous cellular PI3K and tethers it to Dlg1 to form a ternary complex in cells. Extracts (300 mg of protein) of vector, wtORF1, V125A, and T123D cells were immunoprecipitated with either p110a antibody or Dlg1 antibody as indicated. Recovered proteins, as well as cell extract (input), were analyzed in an immunoblot assay with antibodies to the indicated proteins. Note that V125A and T123D cells express Ad9 E4-ORF1 PBM mutants unable to bind PDZ proteins. doi:10.1371/journal.ppat.1004102.g005 undetermined viral factor, which differs from E4-ORF1 by inducing weaker elevation of p85b protein levels and not activating PI3K.
Two carboxyl-terminal residues of E4-ORF1 determine binding to both PI3K and Dlg1 To identify residues of E4-ORF1 required for binding to PI3K, we generated MCF10A lines expressing 16 different E4-ORF1 mutants. We then IPed p110a from lysates of these cell lines, and tested for co-IP of E4-ORF1 (data not shown). One E4-ORF1 mutant, mutant KI, was defective for binding to PI3K compared to the wt E4-ORF1 protein ( Figure 7A, compare lanes 6 and 7). Mutant KI has two point mutations that change the adjacent carboxyl-terminal residues K120 and I121 to alanine residues ( Figure 7B) [5]. We also IPed Dlg1 from the lysates and tested for co-IP of E4-ORF1. The data showed that mutant KI is similarly defective for binding to Dlg1 ( Figure 7A, compare lanes 9 and 10), consistent with a previous report [5]. These binding defects of mutant KI are specific and do not reflect general protein misfolding or complete loss of function because it retains the capacity to form homo-trimers and to bind the cellular PDZ proteins MUPP1, MAGI-1, and ZO-2 [5]. Thus, we identified two E4-ORF1 residues that specifically determine both interactions required to form the ternary complex. Interestingly, the KI residues are located at the extreme carboxyl-terminus near the PBM required for binding to Dlg1, as well as to other PDZ proteins, and additionally overlap the TRI element required for E4-ORF1 homo-trimerization ( Figure 7B).
Dlg1 in the ternary complex functions to recruit both E4-ORF1 and PI3K to the plasma membrane The requirement for Dlg1 to support E4-ORF1-induced PI3K activation correlates with its ability to recruit E4-ORF1 protein to the plasma membrane [34]. This observation, together with  . Two adjacent residues located at the E4-ORF1 carboxyl-terminus determine binding to both PI3K and Dlg1. (A) E4-ORF1 mutant KI is defective in binding to both PI3K and Dlg1. Co-IP assays were conducted with the indicated MCF10A lines as described in the legend to Figure 5. Vertical lines denote removal of extraneous sample lanes. (B) The location of the KI residues at the extreme carboxyl-terminus of E4-ORF1, along with their close proximity to the PBM and overlap with the TRI element, is shown. Also illustrated is that the KI residues determine PI3K binding; both the KI residues and the PBM element, defined by residues TLV, determine Dlg1 binding; and the TRI element, defined by residues VKI, determines E4-ORF1 homotrimerization. doi:10.1371/journal.ppat.1004102.g007 discovery of the Dlg1:E4-ORF1:PI3K ternary complex, led us to hypothesize that Dlg1 recruits not only E4-ORF1 but also PI3K to the plasma membrane. This hypothesis predicts that E4-ORF1 and PI3K would co-localize with Dlg1 at the plasma membrane in wtORF1 cells, but not in V125A cells where the PBM mutant V125A protein can neither bind Dlg1 nor activate PI3K, yet can bind PI3K ( Figure 5). We investigated this idea in indirect immunofluorescence (IF) assays where vector, wtORF1, and V125A cells were double labeled with antibodies to Dlg1 and either E4-ORF1 or p85 and then analyzed by confocal microscopy.
We first examined each cell line for co-localization of E4-ORF1 and Dlg1 (Figure 8). In wtORF1 cells, E4-ORF1 protein localized in the cytoplasm, exhibiting both diffuse and punctate distributions, as well as at the plasma membrane. Previous findings indicated that the cytoplasmic punctae reflect a combination of E4-ORF1 monomer sequestration of TJ-associated PDZ proteins and E4-ORF1 trimer association with membrane vesicles whereas the plasma membrane fraction represents E4-ORF1 trimer binding to Dlg1 [5,34]. Consistent with these findings, the PBM mutant V125A protein failed to localize at the plasma membrane, but retained the cytoplasmic punctate distribution, likely through the PBM-independent association of E4-ORF1 with membrane vesicles [15]. Unlike the wt E4-ORF1 protein, the mutant V125A protein also accumulated in the nucleus, a defect of PBM mutants attributed to passive nuclear diffusion resulting from loss of PBMdependent anchoring to PDZ proteins at extranuclear sites [15]. We also note that in IF assays described here and below, V125A and T123D cells yielded identical results (data not shown). More importantly, while we found that Dlg1 similarly localized at cellcell contact regions of the plasma membrane in vector, wtORF1, and V125A cells, only the plasma membrane-associated E4-ORF1 protein fraction in wtORF1 cells co-localized with Dlg1. Quantified IF data for this experiment, and other IF experiments detailed below, are presented in Table S9.
We next tested the cell lines for co-localization of p85 and Dlg1 ( Figure 9). In vector and V125A cells, p85 exhibited a cytoplasmic distribution but, due to an absence at the plasma membrane, it failed to co-localize with Dlg1. In wtORF1 cells, p85 was similarly distributed in the cytoplasm and, more importantly, was additionally detected at the plasma membrane where it co-localized with Dlg1. Furthermore, the latter effect was decreased by shRNA-mediated Dlg1 depletion in wtORF1 cells ( Figure 10). These data, together with those presented in Figure 8, indicated that PBM-mediated binding of the wt E4-ORF1 protein to Dlg1 functions not only to assemble the Dlg1:E4-ORF1:PI3K ternary complex but also to localize PI3K to the plasma membrane.
The ternary complex stimulates PI3K signaling at the plasma membrane To obtain evidence that the membrane-associated Dlg1:E4-ORF1:PI3K ternary complex mediates PI3K signaling, we investigated whether P-Akt, which is recruited to membrane sites of PI3K activation by binding to PI3K product PIP3, also colocalizes with Dlg1 at the plasma membrane ( Figure 11). Consistent with results of immunoblot assays shown in Figures 2  and 5, P-Akt staining was detected in wtORF1 cells but not in vector or V125A cells. In wtORF1 cells, P-Akt distributed in a speckled pattern in the cytoplasm, and also accumulated at the plasma membrane where it co-localized with Dlg1. We observed a similar accumulation of total Akt protein at the plasma membrane of wtORF1 cells but not vector or V125A cells ( Figure S3). In addition, this accumulation of P-Akt at the plasma membrane was diminished by shRNA-mediated Dlg1 depletion in wtORF1 cells ( Figure 12). These data supported the conclusion that the ternary complex mediates PI3K signaling at the plasma membrane. Figures 8 and 9 demonstrated that both E4-ORF1 and PI3K are recruited to Dlg1 located at the plasma membrane of MCF10A cells. We previously reported that E4-ORF1 also induces cytoplasmic Dlg1 to translocate to the plasma membrane [34]. This finding led to the expectation that wtORF1 cells would show higher Dlg1 membrane staining and lower Dlg1 cytoplasmic staining than vector or V125A cells. While this effect was evident in Figures 10 and 12, it was either weak or absent in Figures 8, 9 and 11. These variable results can be explained by the fact that prior to formation of adherens junctions, Dlg1 localizes primarily in the cytoplasm whereas during formation of adherens junctions, Dlg1 increasingly concentrates at this site of Figure 8. Dlg1 in the ternary complex recruits E4-ORF1 to the plasma membrane. In these indirect immunofluorescence assays, the indicated MCF10A lines were dually stained with E4-ORF1 (green) and Dlg1 (red) antibodies, followed by visualization by fluorescence confocal microscopy, as described in the Materials and Methods. Nuclei were counterstained with DAPI (blue). Individual and merged images are shown. White scale bar = 20 mm. doi:10.1371/journal.ppat.1004102.g008 the plasma membrane, even in the absence of E4-ORF1. In experiments where the E4-ORF1 effect was weak or absent, the cells were post-confluent and had concentrated Dlg1 at mature adherens junctions, thereby partially or completely masking E4-ORF1-induced Dlg1 membrane recruitment. Thus, to optimize detection of this E4-ORF1 activity, we compared the localization of Dlg1 in confluent vector, wtORF1, and V125A cells prior to formation of adherens junctions ( Figure 13). Under these conditions, wtORF1 cells displayed higher Dlg1 membrane staining and lower cytoplasmic staining than vector or V125A cells. This finding demonstrated that E4-ORF1 also promotes cytoplasmic Dlg1 to translocate to the plasma membrane of MCF10A cells.

The ternary complex is required for E4-ORF1-induced transformation of human epithelial cells
We next investigated the functional importance of the ternary complex in cells. The capacity of cells to form colonies when suspended in soft agar measures anchorage-independent growth, a property that correlates best with tumorigenic potential [40]. Previous studies showed that Ad9 E4-ORF1-expressing rodent fibroblasts and Ad9-induced mammary tumor cells form colonies in soft agar and that this oncogenic property depends on E4-ORF1-induced PI3K activation [7,34]. In soft agar assays conducted with the MCF10A lines, we found that wtORF1 cells formed colonies with a high cloning efficiency (95%61.5% SD; n = 3 independent experiments), whereas V125A and T123D cells behaved like vector cells by not forming colonies ( Figure 14A). Moreover, the PI3K inhibitor LY abolished colony formation by wtORF1 cells (Figure 14B). Transformation assays measuring focus formation by these MCF10A lines yielded similar results ( Figures 14C and 14D). To implicate Dlg1 directly in E4-ORF1-induced cellular transformation, we compared colony formation by wtORF1 cells expressing the Dlg1 shRNA and control wtORF1 cells expressing the scrambled Dlg1 shRNA (see Figure 3A, lanes 3 and 4). Initial experiments, however, failed to detect lower colony formation by the Dlg1 shRNA-expressing   (Table S2) and the Dlg1 shRNA reduces these levels by only 3.2-to 3.6-fold (Table S4), we considered the possibility that the Dlg1 shRNA-mediated reduction in P-Akt levels may be insufficient to decrease colony formation. To test this idea, we performed the soft agar assays in the presence of the PI3K inhibitor drug LY at the sub-inhibitory dose of 25 mM, which is one-fourth the concentration required to abolish PI3K activation, soft agar growth, and focus formation in MCF10A cells (Figures 2, 14B, and 14D). Under these conditions, colony formation by wtORF1 cells expressing the Dlg1 shRNA was substantially reduced compared to control cells ( Figure 14E), consistent with previous findings [34]. Immunoblot assays confirmed that, when cultured with 25 mM LY, wtORF1 cells expressing the Dlg1 shRNA have lower levels of Dlg1 and P-Akt and but not E4-ORF1 than the control cells ( Figure 14F). Collectively, our findings supported the conclusion that both the Dlg1:E4-ORF1:PI3K ternary complex and its stimulation of PI3K signaling are essential for the function of E4-ORF1 in cells. The results additionally showed that the wt E4-ORF1 protein is capable of promoting transformation of a non-transformed human epithelial cell line.

Discussion
The human adenovirus E4-ORF1 protein mediates constitutive activation of cellular PI3K, a function shown to promote  tumorigenesis in experimental animals and to augment viral replication, prolong survival, and modulate lipid and glucose metabolism in cells [7][8][9][10][11]. While it is known that PI3K activation by E4-ORF1 depends on its interaction with the cellular PDZ protein Dlg1 and on localization of the resulting Dlg1:E4-ORF1 complex to the plasma membrane, the underlying molecular mechanism for activation had not been previously determined. We report here the first mechanistic insight into this activity by identifying the PI3K p85:p110 heterodimer as a new cellular target of the E4-ORF1 protein.
Based on results reported here along with previously published findings, the mechanism for E4-ORF1-mediated PI3K activation shown in Figure 15 has emerged. We propose that in step 1, E4-ORF1 via the KI residues binds directly to PI3K to form a cytoplasmic E4-ORF1:PI3K heterocomplex (Figures 1, 5, and  7). In step 2, E4-ORF1 within this heterocomplex also interacts with Dlg1-I3 ( Figure 4) [34], via cooperative binding between two PBM+KI elements in the E4-ORF1 trimer and two PDZ domains in Dlg1 (Figure 7) [5,34], thereby assembling a Dlg1:E4-ORF1:PI3K ternary complex (Figures 5 and 6A). Our previous finding that only homo-trimeric E4-ORF1 can interact with Dlg1-I3 [5], which is crucial for E4-ORF1-induced PI3K activation ( Figures 3A and 4) [34], implies that PI3K likewise binds to the E4-ORF1 trimer in both the ternary complex and the E4-ORF1:PI3K heterocomplex, though it remains possible that the E4-ORF1 monomer also binds PI3K. More importantly, a key consequence of assembling the ternary complex is recruitment of the cytoplasmic E4-ORF1:PI3K heterocomplex to the plasma membrane in a Dlg1-dependent fashion (Figures 8-10 and 13) [34]. Similar to growth factor receptor-and ras-mediated PI3K activation [12], the ternary complex additionally stimulates PI3K catalytic activity (Figure 4) [7]. An interesting possibility is that direct binding of PI3K to both E4-ORF1 and Dlg1 in the ternary complex contributes to the latter effect, as well as to PI3K protein elevation discussed later. In step 3, the increased catalytic activity of PI3K in the ternary complex together with its location at the plasma membrane brings the activated PI3K enzyme into contact with lipid substrate PIP2 to produce PIP3, which in turn recruits Akt to the plasma membrane ( Figure S3), resulting in its activation by phosphorylation (Figures 2, 3A, and 11-12). The ensuing dysregulated activation of Akt and its downstream effectors enhance viral replication and cellular metabolism and survival, and can also promote cellular transformation (Figure 14) [7][8][9]11,34]. Also worth mention is that unpublished results suggest that the proposed mechanism of PI3K activation by Ad9 E4-ORF1 protein is shared by E4-ORF1 proteins encoded by other adenovirus serotypes and subgroups (MK and KK, manuscript in preparation).
Numerous viruses encode proteins that dysregulate PI3K in cells [14,[41][42][43][44][45][46][47][48][49][50][51], though molecular mechanisms are lacking in many cases. When mechanistic details are available, they often involve direct or indirect viral protein:PI3K interactions mediated by phosphorylated tyrosine residues on the viral protein itself or on an activated cellular receptor bound to the viral protein. Examples include mouse polyomavirus middle T antigen and EBV LMP1 and LMP2A that directly bind PI3K by mimicking tyrosine phosphorylated, activated membrane receptors; HBV X, HIV-1 Nef, and HCV NS5 that directly couple PI3K to non-receptor tyrosine kinases; and HPV E5 that indirectly binds to PI3K through an interaction with activated, cellular receptor protein tyrosine kinases [14]. These examples underscore novel differences in PI3K activation by Ad9 E4-ORF1, including the requirement for Dlg1 (Figures 3 and 4) and the lack of association of PI3K with tyrosine-phosphorylated proteins [34].
Our data indicated that the ternary complex not only activates PI3K but also elevates p85/p110 protein levels (Figures 2, 3A, 4,  and 7). The latter effect may be important as PI3K overexpression is reported to dysregulate PI3K signaling. For example, an amplified and over-expressed PIK3CA gene coding for the PI3K catalytic subunit hyper-activates PI3K signaling in several different types of human cancers [52], and an over-expressed wt PIK3CA cDNA in MCF10A cells increases PI3K signaling and cellular proliferation, and provokes cellular transformation [53]. Because treatment of E4-ORF1-expressing cells with a PI3K inhibitor for 30 min, or 2 h (data not shown), failed to diminish p85/p110 levels yet lowered Akt and ERK levels (Figure 2), it seems unlikely that p85/p110 elevation results from the capacity of the downstream PI3K effector complex mTORC1 to increase protein synthesis [52]. We instead postulate that the ternary complex directly mediates Dlg1-dependent p85/p110 protein stability. Results showed that both p85/p110 protein elevation and PI3K activation in wtORF1 cells are diminished by shRNA-mediated Dlg1 depletion or are either severely impaired or absent in V125A and T123D cells (Figures 2 and 3A), revealing a common dependence on Dlg1. Other data showed that the Dlg1-I2 isoform does not support E4-ORF1-induced PI3K activation but does support E4-ORF1-induced PI3K protein elevation (Figure 4), indicating that these two Dlg1-mediated activities are separable. This observation hints to the possible existence of separate pools of ternary complexes containing either Dlg1-I3 or Dlg1-I2. In this scenario, the Dlg1-I2 ternary complex may localize in the cytoplasm and function solely to elevate PI3K protein levels, whereas the Dlg1-I3 ternary complex localizes both in the cytoplasm and at the plasma membrane, with the cytoplasmic complex also functioning to elevate PI3K protein levels and the membrane complex functioning specifically to activate PI3K. Future studies will test these ideas as well as determine whether E4-ORF1-mediated p85/p110 elevation results from increased mRNA levels, protein synthesis, or protein half-life. Results of soft agar and focus formation assays implicated the ternary complex and its activation of PI3K in E4-ORF1-induced transformation of a human epithelial line that retains features of normal epithelial cells ( Figure 14). This finding reinforces concerns that the use of adenovirus vectors retaining the E4-ORF1 gene for vaccination or various therapies, as well as the proposed use of the Ad36 E4-ORF1 gene to treat fatty liver disease and liver dysfunction or to improve glycemic control [11], may increase patient risk for developing neoplasms.
Evidence also indicates that Dlg1 functions to regulate normal PI3K signaling in cells. For example, Laprise et al. showed that Dlg1 is phosphorylated on tyrosine residues at the AJ of human intestinal epithelial cells and that these residues mediate direct binding to and activation of PI3K to promote cellular differentiation [54]. Furthermore, the lipid phosphatase and important human tumor suppressor protein PTEN [13], which antagonizes PI3K signaling by dephosphorylating PIP3, is known to bind Dlg1. This interaction, mediated by the carboxyl-terminal PBM of PTEN and PDZ domain 2 of Dlg1 [55,56], enhances PTEN activity to block proliferation and viability of MCF-7 breast carcinoma cells and to suppress Schwann cell myelination of peripheral nerves [57,58]. In addition to human adenovirus, several other pathogenic human viruses code for PBM-containing proteins that target Dlg1 and also bind to and/or activate PI3K, including the E6 oncoprotein of high-risk HPVs [42,59,60], the Tax oncoprotein of HTLV-1 [61][62][63][64][65], and the NS1 protein of influenza A viruses [66]. These observations suggest that, under normal physiological conditions, Dlg1 functions as a key regulator of PI3K signaling and that pathogenic human viruses commonly hijack this cellular PDZ protein, at least in part, to dysregulate the PI3K pathway and, in doing so, enhance viral infections associated with acute and chronic human diseases and cancer. Hence, studies of human adenovirus and its subversion of cellular PI3K, Dlg1, and other PDZ proteins may yield mechanistic insights that aid development of new therapeutic strategies for treating viral diseases in people.

Cells and viruses
Human MCF10A mammary epithelial cells (American Type Culture Collection) were maintained, as described [39], in complete medium consisting of DMEM/F-12 supplemented with 5% horse serum (Invitrogen, Carlsbad, CA), 20 ng/ml epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ), 100 mg/ml hydrocortisone, 10 mg/ml insulin, 1 ng/ml cholera toxin, and 20 mg/ml gentamicin (Sigma-Aldrich, St. Louis, MO). MCF10A lines were generated by transduction with retroviral vector pBABE and/or pSUPER-retro followed by selection in complete medium containing 2 mg/ml puromycin and/or 25 mg/ml blasticidin. Experiments utilized pools of selected cells passaged five times or less, except for the experiment presented in Figure 12B, which used cells at passage number 8. The same numbers of cells were plated for all experiments comparing different cell lines or treatments. For some experiments, cells were passaged into complete medium containing a lower concentration of EGF (5 ng/ml) [39]. PI3K inhibitor LY294002 was purchased (Cell Signaling Technology, Inc., Beverly, Massachusetts). Transfections were performed with TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI). Wt and mutant Ad9 viruses and their propagation in human A549 cells were described [7,16].
For cell fractionation assays [15], cells were lysed in RIPA buffer and then centrifuged (10,0006g for 15 min at 4uC) to separate the detergent-soluble supernatant and detergent-insoluble pellet fractions. The pellet fraction was subsequently solubilized in a volume of 26 sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.2 M DTT, 20% glycerol, 0.001% bromophenol blue) equal to that of the detergent-soluble supernatant fraction. Experiments compared equal volumes of the detergent-soluble supernatant and detergentinsoluble pellet fractions.
Pulldowns, immunoprecipitations, immunoblots, and mass spectrometry GST pulldowns and immunoprecipitations with glutathionesepharose beads or protein G-sepharose beads (GE Healthcare Life Sciences), respectively, were carried out as described [4,18]. Recovered proteins and cell extract (30 mg of protein) were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted as described [4,18]. Immunoblotted membranes were imaged with a UVP Biospectrum 810 Imaging System (Upland, CA) and analyzed with VisionWorksLS software. Differences in the levels of specified proteins between two cell samples were quantified by comparing protein band intensities normalized to actin, and the Student's t-test was performed to determine statistical significance. E4-ORF1-binding proteins were identified by conducting a pulldown assay with extracts of suspension-cultured HeLa cells, resolving recovered proteins by SDS-PAGE, digesting separate gel sections with trypsin, and subjecting the released peptides to MALDI-TOF mass spectrometry.

Indirect immunofluorescence and confocal microscopy
Glass slides (Millicell EZ SLIDE, Millipore) were coated with poly-L-lysine (Sigma-Aldrich). Cells plated on the slides were fixed in 2% formaldehyde (Polysciences, Inc.), permeabilized with 0.5% Triton X-100, quenched with 100 mM glycine, blocked in 10% goat serum, and incubated with primary antibody and then with Alexa Fluor 488-conjugated goat anti-rabbit IgG and/or Alexa Fluor 594-conjugated goat anti-mouse IgG secondary antibodies (Life Technologies Corp.). Prior or following incubation with primary antibody, cells were washed between each step with either phosphate-buffered saline (PBS) or immunofluorescence buffer (IFB) [7.7 mM sodium azide, 0.1% (w/v) BSA, 0.2% (v/v) Triton X-100, 0.05% (v/v) Tween-20 in PBS], respectively. Coverslips were mounted on slides with SlowFade Gold antifade reagent (Life Technologies Corp.). Cells were analyzed by confocal microscopy with a Nikon A1-Rs inverted laser-scanning microscope and NIS Elements software. The percentage of cells in which specified proteins localized at the plasma membrane was quantified using Image J software (NIH).

Soft agar and focus formation assays
Soft agar assays were carried out as described [3]. Briefly, in complete medium, 3610 5 cells were suspended in 1 ml of 0.4% noble agar (Affymetrix) and placed atop a 2 ml 0.8% noble agar underlay in a 6-well plate. Cells were fed complete medium every other day. Colonies were documented with a Nikon D70S camera mounted on a Nikon TMS inverted microscope. ImageJ software (NIH) was used to score the numbers of cells that (a) did and (b) did not form a colony, and cloning efficiency (a/a+b) was calculated from .300 scored cells per experiment. For focus formation assays, 300 wtORF1, V125A, or vector cells mixed with 3610 5 vector cells in complete medium were seeded into a 6-well plate. Cells were fed every 3 days and, when visible, foci were stained with crystal violet (5 mg/ml in 25% methanol) and photographed with a Canon PowerShot A1200 digital camera. ANOVA with Tukey post-hoc analysis was performed to determine statistical significance.

Statistical analyses
Microsoft Excel was used to calculate the mean, standard deviation (SD), and standard error of the mean (SEM) for data. R statistical software was used to determine statistical significance. Standard denotation of asterisks for p values was used (*, p,0.05; **, p,0.01; ***, p,0.001).  Figure S1 The vast majority of E4-ORF1 protein is contained within the insoluble pellet fraction of MCF10A cells. Soluble (S) and insoluble (I) extract fractions of vector and wtORF1 cells were prepared (see Materials and Methods). An equivalent amount of each fraction was analyzed in an immunoblot assay with E4-ORF1 antibody. The soluble fraction was additionally immunoblotted with actin antibody as a loading control. (TIFF) Figure S2 Dominant-negative mutant rasN17 blocks PI3K activation by mutant rasV12 but not E4-ORF1. Mutant rasN17 (A) blocks mutant rasV12-induced PI3K activation but (B) has no effect on E4-ORF1-induced PI3K activation. MCF10A cells were transfected with expression plasmid GW1-rasN17 (2 mg), GW1-E4-ORF1 (75 ng), or GW1-rasV12 (500 ng) alone or in the indicated combinations. The total amount of DNA in each transfection was equalized to 7.575 mg using empty GW1 plasmid. At 48 h post-transfection, cells were serum starved for 1 h, and then extracts were prepared and analyzed in immunoblot assays using antibodies to the indicated proteins. The insoluble (I) pellet fraction (see Materials and Methods) was immunoblotted for E4-ORF1 protein. Vertical lines in (A) denote removal of extraneous sample lanes. (TIFF) Figure S3 Total Akt accumulates at the plasma membrane in wtORF1 cells. In IF assays, the indicated MCF10A lines stained with an antibody reactive to total Akt (green) were visualized by fluorescence confocal microscopy. Nuclei were counterstained with DAPI (blue). Individual and merged images are shown. White scale bar = 20 mm.

(TIFF)
Table S1 Cellular proteins identified to interact with GST-E4-ORF1 in a pulldown assay. Shown are 10 selected proteins identified by mass spectrometry to bind the Ad9 E4-ORF1 fusion protein in a pulldown assay conducted with extracts from HeLa cells.   Figure 6A, average fold changes in levels of the indicated proteins were quantified from independent immunoblots of Ad9-V125A virus-infected cells versus mockinfected cells. See Materials and Methods for details. (DOCX)