hijacking discs large 1 for oncogenic phosphatidylinositol 3...

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1 Hijacking Discs Large 1 for Oncogenic Phosphatidylinositol 3-Kinase 2

Activation in Human Epithelial Cells Is a Conserved Mechanism of 3 Human Adenovirus E4-ORF1 Proteins 4

Manish Kumar, Kathleen Kong, and Ronald T. Javier# 5 Department of Molecular Virology and Microbiology, Baylor College of Medicine, 6

Houston, Texas, United States of America 7 8 9 10 Running Title: Conserved Mechanism of PI3K Activation by E4-ORF1 11 #Address correspondence to Ron Javier, [email protected] 12 Word counts: 13

Abstract - 211 14 Importance - 121 15 Text - 5374 16

JVI Accepts, published online ahead of print on 24 September 2014J. Virol. doi:10.1128/JVI.02324-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 17 The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes 18 viral replication as well as cellular metabolic reprogramming, survival, and 19 transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). 20 We recently reported that the E4-ORF1 protein from subgroup D human adenovirus 21 type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving 22 separate interactions of E4-ORF1 with cellular Discs large 1 (Dlg1) and PI3K to form a 23 ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. 24 Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, 25 doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate 26 whether other human adenovirus E4-ORF1 proteins share this mechanism of action. 27 Results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 28 proteins encoded by representative human adenovirus serotypes from subgroups A 29 to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein 30 elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular 31 transformation. The first three of these E4-ORF1 activities were also observed in 32 MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. 33 Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a 34 conserved molecular mechanism of PI3K activation, which confers a common capacity 35 to promote oncogenic transformation in human epithelial cells. 36


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IMPORTANCE 37 PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular 38 transformation by human adenovirus type 9, augments viral protein expression and 39 replication by human adenovirus type 5, and dysregulates cellular glucose and lipid 40 metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 41 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular 42 mechanism to mediate constitutive PI3K activation that can provoke oncogenic 43 transformation in human epithelial cells. The results raise potential safety concerns 44 about the use of vectors encoding the E4-ORF1 gene in human gene therapy and 45 vaccination. Our findings further suggest that the conserved mechanism revealed here 46 may be targeted for development of therapeutic drugs to treat and prevent adenovirus-47 associated human diseases. 48


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INTRODUCTION 49 The >60 known serotypes of human adenovirus are classified into seven 50

subgroups (A to G) based on hemagglutination properties, oncogenicity in rodents, DNA 51 homology, and genomic organization (1). In people, these viruses cause a variety of 52 acute diseases by infecting epithelial cells that line mucous membranes (1). 53 Furthermore, replication-defective adenovirus vectors are common vehicles for human 54 gene therapy and vaccination. The oncogenic potential of certain adenoviral genes has 55 been studied to reveal molecular mechanisms involved in the development of human 56 cancers (2). 57

Human adenovirus type 9 (Ad9) is a member of subgroup D that consists of 58 viruses primarily associated with eye infections in people. In experimentally infected 59 rats, however, Ad9 elicits estrogen-dependent mammary tumors, and the viral E4-ORF1 60 gene is the major oncogenic determinant (3-5). Adenovirus E4-ORF1 evolved from 61 cellular dUTPase (6), which encodes a conserved enzyme of nucleotide metabolism. 62 Although E4-ORF1 and dUTPase are predicted to share a common protein fold, they 63 have functionally diverged as evidenced by E4-ORF1’s lack of dUTPase catalytic 64 activity (6, 7). Instead, E4-ORF1 dysregulates cellular class IA phosphatidylinositol 65 3-kinase (PI3K) (8). This conserved E4-ORF1 activity is critical for mammary 66 tumorigenesis and cellular transformation by Ad9 (8), optimal replication of human 67 adenovirus type 5 (Ad5) (9, 10), promotion of cell survival by an Ad5 vector (11), and 68 reprogramming of cellular lipid and glucose metabolism by human adenovirus type 36 69 (Ad36) (12). 70

Composed of p85 regulatory and p110 catalytic subunits, PI3K is a lipid kinase 71


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and key downstream effector of membrane receptors and ras. In their activated states, 72 membrane receptors and ras recruit PI3K to the plasma membrane to stimulate 73 conversion of the PI3K lipid substrate phosphatidylinositol 4,5-bisphosphate (PIP2) to 74 phosphatidylinositol 3,4,5-trisphosphate (PIP3) (13). PIP3 acts as a second messenger 75 to recruit PI3K effector proteins such as Akt to the plasma membrane, where Akt 76 becomes activated by PDK1- and mTORC2-mediated phosphorylation on threonine 308 77 (T308) and serine 473 (S473), respectively. Akt downstream effectors control critical 78 cellular processes such as metabolism, protein synthesis, growth, survival, migration, 79 and proliferation. Notably, dysregulation of PI3K also plays a central role in human 80 disease, including cancers and infections caused by viruses, which commonly subvert 81 the PI3K signaling pathway to enhance viral replication and virus-host interactions (14, 82 15). 83

PI3K activation induced by Ad9 E4-ORF1 requires its interaction with the cellular 84 PDZ protein Dlg1. Dlg1 mediates recruitment of the resulting Dlg1:E4-ORF1 complex to 85 the plasma membrane (16). In a recent study, we exposed the mechanism of 86 Ad9 E4-ORF1-induced PI3K activation by demonstrating that E4-ORF1 within the 87 Dlg1:E4-ORF1 complex additionally binds directly to PI3K, resulting in formation of the 88 Dlg1:E4-ORF1:PI3K ternary complex that translocates PI3K to the plasma membrane 89 (17). Our study also showed that the ternary complex upregulates the PI3K 90 p85 regulatory and p110 catalytic protein subunits and promotes PI3K-dependent 91 oncogenic cellular transformation. 92

E4-ORF1 proteins from subgroups A to D display 45-51% amino-acid identity, 93 65-69% amino-acid similarity, and a conserved capacity to activate PI3K (6, 8). The 94


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goal of the current study was to determine whether other E4-ORF1 proteins share the 95 novel mechanism of PI3K activation discovered for subgroup D Ad9 E4-ORF1. We 96 found that in human epithelial cells, E4-ORF1 proteins from subgroups A to D induce 97 ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, 98 Dlg1 and PI3K recruitment to the plasma membrane, and PI3K-dependent oncogenic 99 cellular transformation. The findings suggest that most, if not all, human adenovirus 100 E4-ORF1 genes share a conserved mechanism of PI3K dysregulation that can provoke 101 oncogenic transformation in human epithelial cells. 102


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MATERIALS AND METHODS 103 Plasmids and antibodies. The pBABE-puro or -neo plasmids containing a cDNA for 104 rasV12 or wt Ad9 E4-ORF1 and the pSUPER-retro plasmids encoding the Dlg1 shRNA 105 and matched scrambled shRNA were described (17). The cDNAs of wt or HA-tagged 106 E4-ORF1 from Ad12, Ad3, Ad5, Ad9, and Ad36 were cloned into pBabe-puro using 107 either the BamHI and EcoRI sites or the EcoRI site, respectively. Ad9 E4-ORF1 108 antiserum was described (4). Antibodies to p110α, Akt, phospho-Akt(Ser473), 109 phospho-Akt(Thr308), p42/44 MAPK, and phospho-p42/44 MAPK (Thr202/Tyr204) 110 (Cell Signaling Technologies), to p85β, SAP97 (Dlg1), (Santa Cruz Biotechnologies), to 111 p85α, p85α/β, and actin (Millipore), to ras (BD Biosciences), and to HA (Sigma-Aldrich) 112 were purchased. 113 Cells, retroviral vectors, and viruses. Human MCF10A mammary epithelial cells 114 (ATCC) are an immortalized but non-transformed mammary epithelial cell line having 115 properties of normal breast cells (18). MCF10A cells were maintained in complete 116 medium consisting of DMEM/F-12 supplemented with 5% horse serum (Invitrogen, 117 Carlsbad, CA), 20 ng/ml epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ), 118 100 µg/ml hydrocortisone, 10 µg/ml insulin, 1 ng/ml cholera toxin, and 20 µg/ml 119 gentamicin (Sigma-Aldrich, St. Louis, MO). Transfections of Phoenix-ampho cells 120 (ATCC) to produce retroviral vectors were performed with TransIT-LT1 Transfection 121 Reagent (Mirus Bio, Madison, WI). MCF10A lines were generated by transduction with 122 retroviral vector pBABE and/or pSUPER-retro followed by selection in complete medium 123 containing 2 µg/ml puromycin and/or 500 µg/ml G418. Experiments used pools of 124 selected cells passaged five times or less. The same numbers of cells were plated for 125


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each experiment comparing different cell lines or treatments. For experiments, cells 126 were passaged into complete medium containing a lower concentration of EGF 127 (5 ng/ml). PI3K inhibitor LY294002 was purchased (Cell Signaling Technology, Inc., 128 Beverly, MA). Wt Ad12, Ad3, Ad5 and Ad9 viruses were propagated and titrated in 129 human A549 cells as described previously (19). 130 Cell extracts, immunoprecipitations, and immunoblots. Extracts were prepared, as 131 described (20), by lysis of cells in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl 132 pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors 133 (2 mM PMSF, 20 µg/ml each of leupeptin and aprotinin) and phosphatase inhibitors 134 (50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate). Protein 135 concentrations were determined using the BCA protein estimation kit 136 (Pierce Biotechnology, Inc, Rockford, IL). Immunoprecipitations with protein 137 G-sepharose beads (GE Healthcare Life Sciences) were carried out as described (21). 138 Recovered proteins and cell extract (30 µg of protein) were resolved by SDS-PAGE, 139 transferred to a PVDF membrane, and immunoblotted as described (21). 140 Immunoblotted membranes were imaged with a UVP Biospectrum 810 Imaging System 141 (Upland, CA) and analyzed with VisionWorksLS software. Differences in protein levels 142 between two samples were quantified by comparing each band signal normalized to the 143 corresponding actin band signal. 144 Indirect immunofluorescence and confocal microscopy. Glass slides (Millicell 145 EZ SLIDE, Millipore, Billerica, MA) were coated with poly-L-lysine (Sigma-Aldrich, 146 St. Louis, MO). Cells plated on the slides were fixed in 2% formaldehyde (Polysciences, 147 Inc., Warrington PA), permeabilized with 0.5% Triton X-100, quenched with 100 mM 148


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glycine, blocked in 10% goat serum (Sigma-Aldrich, St. Louis, MO), and incubated with 149 primary antibody and then with Alexa Fluor 488-conjugated goat anti-rabbit IgG and/or 150 Alexa Fluor 594-conjugated goat anti-mouse IgG secondary antibodies 151 (Life Technologies Corp. Grand Island, NY). Before or after incubation with primary 152 antibody, cells were washed between each step with either phosphate-buffered saline 153 (PBS) or immunofluorescence buffer (IFB) [7.7 mM sodium azide, 0.1% (w/v) BSA, 154 0.2% (v/v) Triton X-100, 0.05% (v/v) Tween-20 in PBS], respectively. Coverslips were 155 mounted on slides with SlowFade Gold antifade reagent containing DAPI (Life 156 Technologies Corp. Grand Island, NY). Cells were analyzed by confocal microscopy 157 with a Nikon A1-Rs inverted laser-scanning microscope using NIS Elements software. 158 The percentage of cells in which specified proteins localized at the plasma membrane 159 was quantified pictures using Image J software (NIH). 160 Soft agar assays. Soft agar assays were carried out as described (4). Briefly, in a 161 6-well plate, 3.3 X 105 cells were suspended in 1 ml of complete medium containing 162 0.45% noble agar (Affymetrix, Santa Clara, CA) and placed atop 2 ml of a complete 163 medium underlay containing 0.8% noble agar. Cells were fed with complete medium 164 that was replaced every other day. Colony formation was documented with a Nikon 165 D70S camera mounted on a Nikon TMS inverted microscope. In pictures, 166 ImageJ software (NIH) was used to score the numbers of cells that (a) did and (b) did 167 not form a colony, and cloning efficiency (a/a + b) was calculated from >250 scored 168 cells per experiment. 169 Statistical analyses. Microsoft Excel was used to calculate the mean, standard 170 deviation (SD), and standard error of the mean (SEM). The Student's t-test was used to 171


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determine the statistical significance of results. Standard denotation of asterisks for 172 p values was used.173


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RESULTS 174 E4-ORF1 proteins from human adenovirus subgroups A to D activate PI3K and 175 upregulate the PI3K catalytic and regulatory protein subunits. We initially sought 176 to determine whether, like Ad9 E4-ORF1 (17), E4-ORF1 proteins encoded by 177 representative adenovirus serotypes from subgroups A to C activate PI3K and elevate 178 its protein levels in human MCF10A epithelial cells (18). To address this question, we 179 generated MCF10A lines stably transduced with an empty pBABE retroviral expression 180 vector (vector cells) or with pBABE encoding either the wild-type (wt) or amino-terminal 181 HA-epitope tagged E4-ORF1 gene from subgroup A human adenovirus type 12 (Ad12) 182 (12ORF1 or HA-12ORF1 cells), subgroup B human adenovirus type 3 (Ad3) (3ORF1 or 183 HA-3ORF1 cells), subgroup C Ad5 (5ORF1 or HA-5ORF1 cells), or subgroup D Ad9 184 (9ORF1 or HA-9ORF1 cells) (6). As a control, some experiments included an 185 MCF10A line transduced with pBABE encoding the rasV12 oncogene (rasV12 cells) 186 (17). 187

Immunoblots of cell extracts verified rasV12 overexpression in rasV12 cells 188 (Fig. 1A, compare lane 3 to lane 1) and HA-E4-ORF1 expression in HA-E4-ORF1 lines 189 (Fig. 1A, compare lane 7 to lanes 5, 9, and 11; Fig. 2A, lanes 1-5). Expression of 190 HA-3ORF1 was somewhat higher than comparably expressed HA-12ORF1, -5ORF1, 191 and -9ORF1. Although the four HA-E4-ORF1 polypeptides share the same predicted 192 molecular mass of 15-kDa, HA-3ORF1 and -9ORF1 aberrantly migrated at ~20-kDa in 193 protein gels, whereas HA-12ORF1 and -5ORF1 migrated as expected. More 194 importantly, higher levels of activated, phosphorylated Akt (P-Akt) were observed in 195 rasV12 cells and HA-E4-ORF1 lines than in vector cells (Fig. 1A, compare lanes 3, 5, 7, 196


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9, 11 to lane 1). From cumulative experiments with the HA-E4-ORF1 lines, the average 197 increases in P-Akt S473 relative to vector cells ranged from 25- to 45-fold (p ≤ 1.3E-02*). 198 RasV12 cells, but not HA-E4-ORF1 lines, additionally displayed higher levels of 199 activated, phosphorylated MAP kinases Erk-1 and -2 (P-Erk1/2) than vector cells 200 (Fig. 1A, compare lane 3 to lanes 1, 5, 7, 9, 11), consistent with previous findings (16, 201 17). Furthermore, compared to vector cells, HA-E4-ORF1 lines exhibited elevated 202 levels of PI3K protein subunits p110α, p85α, and p85β (Fig. 2A, compare lanes 2-5 to 203 lane 1). From cumulative experiments, the average increases in p110α, p85α, and 204 p85β relative to vector cells ranged from 10- to 18-fold (p ≤ 7.6E-03**), 8.4- to 15-fold 205 (p ≤ 2.2E-02*), and 12- to 27-fold (p ≤ 3.3E-02*), respectively. 206

Experiments similar to those described above with HA-E4-ORF1 lines were also 207 conducted with wt E4-ORF1 lines and yielded comparable results (Fig. 1B and 2B, 208 lanes 1-5), aside from the detection of higher p85β protein levels in 9ORF1 cells than in 209 other wt E4-ORF1 lines (Fig. 1B and 2B, compare lane 5 to lanes 2-4). In 210 wt E4-ORF1 lines (Fig. 1B and 2B), E4-ORF1 expression was only detected in 9ORF1 211 cells because the Ad9 E4-ORF1 antibody does not cross-react with E4-ORF1 proteins 212 from subgroups A to C. In addition, the lower Dlg1 levels detected in HA-E4-ORF1 and 213 wt E4-ORF1 lines relative to vector cells in Fig. 2 were not reproducible as the same 214 lines showed comparable Dlg1 levels in Fig. 1B and 5A. 215

Treatment with the PI3K inhibitor drug LY294002 (LY) returned the high P-Akt 216 levels in rasV12 cells and HA-E4-ORF1 lines to those of vector cells (Fig. 1A, compare 217 lanes 4, 6, 8, 10, 12 to lane 2), indicating that their elevated P-Akt levels resulted from 218 PI3K activation. By contrast, LY treatment elevated P-Erk levels in rasV12 cells and 219


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HA-E4-ORF1 lines (Fig. 1A, compare lanes 3-4, 5-6, 7-8, 9-10, and 11-12), presumably 220 by relieving Akt-mediated inhibition of Raf-induced MAP kinase activation (22). Taken 221 together, data from human epithelial cells showed that E4-ORF1 proteins from 222 subgroups A to D activate PI3K and upregulate PI3K protein subunits p110α, p85α, and 223 p85β, yet do not activate Erk1/2. 224 E4-ORF1 proteins from human adenovirus subgroups A to D promote 225 PI3K-dependent cell growth in soft agar. Colony formation by cells suspended in soft 226 agar measures anchorage-independent growth, a transformed property correlating best 227 with tumorigenic potential (23). The Ad9 E4-ORF1 oncogene induces MCF10A cells to 228 grow in soft agar in a PI3K-dependent manner (17), so we examined the MCF10A lines 229 described above for similar properties. Vector cells did not form colonies in soft agar, 230 whereas rasV12 cells and the HA-E4-ORF1 and wt E4-ORF1 lines did so with high 231 cloning efficiencies, ranging from 90-98% (Fig. 3). Treatment with the PI3K inhibitor LY 232 ablated colony formation by the HA-E4-ORF1 and wt E4-ORF1 lines (Fig. 3). The 233 observation that PI3K inhibition did not ablate colony formation by rasV12 cells indicates 234 that an undetermined ras effector(s), possibly Erk1/2 that was enhanced by LY 235 treatment (Fig. 1A, compare lanes 3 and 4), supports this activity. Therefore, E4-ORF1 236 proteins from subgroups A to D comparably induced PI3K-dependent oncogenic 237 transformation of human epithelial cells. 238 E4-ORF1 proteins from human adenovirus subgroups A to D form the ternary 239 complex. To determine whether, like Ad9 E4-ORF1 (17), E4-ORF1 proteins from 240 subgroups A to C form the ternary complex (Dlg1:E4-ORF1:PI3K), we 241 immunoprecipitated (IPed) the PI3K catalytic subunit p110α from lysates of vector cells 242


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and HA-E4-ORF1 lines, and tested for co-immunoprecipitation (coIP) of HA-E4-ORF1 243 and Dlg1, as well as p85α and p85β, in immunoblots. As expected, we detected coIP of 244 the PI3K regulatory subunits p85α and p85β with p110α in all cells (Fig. 2A, compare 245 lanes 6-10). More importantly, we showed coIP of both HA-E4-ORF1 and Dlg1 with 246 p110α in HA-E4-ORF1 lines but not vector cells (Fig. 2A, compare lanes 7-10 to lane 6). 247 In a reciprocal assay using the same cell lysates, we IPed Dlg1 and tested for coIP of 248 HA-E4-ORF1, p110α, p85α, and p85β. For HA-E4-ORF1 lines but not vector cells, we 249 observed coIP of HA-E4-ORF1, p110α, and p85β with Dlg1 (Fig. 2A, compare lanes 250 12-15 to lane 11), and also coIP of p85α with Dlg1 specifically from HA-12ORF1 cells 251 (Fig. 2A, compare lane 12 to lanes 11 and 13-15). In addition, comparisons of the 252 p110α and Dlg1 coIP data suggested that ternary complexes from subgroup A to D 253 HA-E4-ORF1 proteins vary somewhat in incorporation of the p85α and p85β PI3K 254 subunits (Fig. 2A, compare lanes 7-10 to lanes 12-15). 255

We next IPed Dlg1 from lysates of the wt E4-ORF1 lines. The data in Fig. 2B 256 showed coIP of p110α, p85α, and p85β with Dlg1 from all wt E4-ORF1 lines but not 257 vector cells (compare lanes 7-10 to lane 6) and, as expected, coIP of Ad9 E4-ORF1 258 with Dlg1 only from 9ORF1 cells (compare lane 10 to lane 6-9). In 9ORF1 cells, the 259 higher levels of p85β were associated with increased coIP of p85β with Dlg1 (Fig. 2B, 260 compare lane 10 to lane 7-9). Furthermore, the more robust association of p85α with 261 Dlg1 observed in wt E4-ORF1 lines than in HA-E4-ORF1 lines suggested that the 262 HA tag weakens the ability of E4-ORF1 proteins from Ad3, Ad5, and Ad9, but less so 263 Ad12, to tether Dlg1 to the p85α:p110α PI3K heterocomplex. Nonetheless, collective 264 results from coIP assays with HA-E4-ORF1 and wt E4-ORF1 lines indicated that 265


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E4-ORF1 proteins from subgroups A to D assemble the Dlg1:E4-ORF1:PI3K ternary 266 complex in human epithelial cells. 267

It was also important to determine whether, like an Ad9 infection (17), infections 268 with human adenoviruses from subgroups A to C promote PI3K activation, PI3K protein 269 upregulation, and Dlg1 protein downregulation, though Dlg1 downregulation in 270 Ad9-infected cells probably is not mediated by E4-ORF1. At 24h post infection (hpi) at 271 an MOI of 1, MCF10A cells infected with Ad12, Ad3, Ad5, or Ad9 exhibited higher levels 272 of P-Akt and PI3K subunits than mock-infected MCF10A cells (Fig. 4A, compare lanes 273 2-5 to lane 1). Furthermore, Dlg1 levels were reproducibly 2- to 4-fold lower in all virus-274 infected cells compared to mock-infected cells (Fig. 4A, compare lanes 2-5 to lane 1; 275 Fig. 5B, compare lanes 3, 5, 7, 9 to lane 1), mirroring previous results with Ad9-infected 276 MCF10A cells (17). Again, we expected to detect E4-ORF1 protein only in Ad9-infected 277 cells due to lack of cross-reactivity of the Ad9 E4-ORF1 antibody with E4-ORF1 proteins 278 from subgroups A to C (Fig. 4A, compare lanes 2-5 to lane 1; Fig. 5B, compare lanes 279 9-10 to lanes 3-8). 280

We also IPed Dlg1 from the cell lysates above to investigate ternary complex 281 formation during viral infections. The data showed coIP of Ad9 E4-ORF1, p110α, and 282 p85β with Dlg1 from virus-infected cells but not mock-infected cells (Fig. 4A, compare 283 lanes 7-10 to lane 6), and also coIP of p85α with Dlg1 from Ad12-, Ad3-, and Ad9-284 infected cells but not from Ad5- or mock-infected cells (Fig. 4A, compare lanes 7, 8, and 285 10 to lanes 6 and 9). As a control, we verified comparable viral infections by showing 286 no detectable cytopathic effect or accumulation of viral major capsid protein hexon at 287 6 hpi and both virus-specific cytopathic effects and comparable hexon accumulation at 288


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24 hpi (Fig. 4B and 4C). These findings support the conclusion that during a viral 289 infection of human epithelial cells, E4-ORF1 proteins from subgroups A-D activate PI3K, 290 elevate PI3K protein levels, and form ternary complexes. 291

Comparisons of Dlg1 immunoprecipitations in Fig. 2B and 4A revealed 292 unexpected differences between wt E4-ORF1 lines and adenovirus-infected cells. In 293 Fig. 2B, ternary complexes in all wt E4-ORF1 lines contained similar amounts of p85α 294 (compare lanes 7-10), but ternary complexes in 9ORF1 cells selectively also contained 295 more p85β (compare lane 10 to lanes 7-9), which showed higher expression in these 296 cells (compare lane 5 to lanes 2-4). By contrast, in Fig. 4A, ternary complexes in both 297 Ad12- and Ad9-infected cells selectively contained more p85β (compare lanes 7 and 10 298 to lanes 8-9), whereas ternary complexes in Ad3-infected cells selectively contained 299 more p85α (compare lane 8 to lanes 7 and 9-10). These differences, however, did not 300 always strictly correlate with the relative p85α and p85β protein levels in infected cells. 301 While the basis for the differences between wt E4-ORF1 lines and virus-infected cells is 302 not understood, we postulate that other viral gene products in virus-infected cells may 303 influence the specific p85 isoform composition of ternary complexes. 304

Another unexpected observation in Fig. 4A was the apparent non-stoichiometric 305 ratios of p110α to p85α and p85β in ternary complexes from different virus-infected 306 MCF10A cells. For example, whereas ternary complexes from all adenovirus-infected 307 cells contained similar amounts of p110α, the amounts of p85α or p85β in the 308 complexes were dissimilar (e.g., compare lanes 7 and 9). We speculate that, in a 309 virus-specific fashion, other viral gene products may selectively displace either p85α or 310 p85β from p110α and E4-ORF1 within the ternary complex. 311


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Dlg1 depletion diminishes PI3K activation by E4-ORF1 proteins from human 312 adenovirus subgroups A to D. Because the ternary complex of Ad9 E4-ORF1 313 mediates PI3K activation (17), we asked whether ternary complexes formed by 314 E4-ORF1 proteins from subgroups A to C share this activity. Our approach was to 315 score for decreased PI3K activation by each E4-ORF1 protein in MCF10A cells 316 depleted of Dlg1 using a short hairpin RNA (shRNA). We therefore stably transduced 317 pSUPER vector encoding either the Dlg1 shRNA or the negative-control scrambled 318 shRNA into the HA-E4-ORF1 lines and vector cells. Dlg1 depletion decreased the high 319 P-Akt levels in HA-E4-ORF1 lines but did not affect the low P-Akt levels in vector cells 320 (Fig. 5A, compare lanes 1-2, 3-4, 5-6, 7-8, and 9-10). From three independent 321 experiments with Dlg1 shRNA-expressing HA-E4-ORF1 lines, the average reductions in 322 P-Akt S473 and Dlg1 relative to matched scrambled shRNA-expressing HA-E4-ORF1 323 lines ranged from 1.8- to 4.6-fold (p ≤ 6.6E-03**) or 2.6- to 3.3-fold (p ≤ 5.1E-03**), 324 respectively. Dlg1 depletion likewise decreased the high P-Akt levels in vector cells 325 infected with Ad12, Ad3, Ad5, or Ad9 at 24 hpi yet did not affect the low P-Akt levels in 326 mock-infected vector cells (Fig. 5B, compare lanes 1-2, 3-4, 5-6, 7-8, and 9-10). 327 Table 1 shows the average reductions in P-Akt S473 and Dlg1 for adenovirus-infected 328 Dlg1 shRNA-expressing vector cells relative to matched adenovirus-infected scrambled 329 shRNA-expressing vector cells from two independent experiments. Taken together, the 330 results indicated that ternary complexes formed by E4-ORF1 proteins from subgroups 331 A to D mediate PI3K activation in either stable E4-ORF1-expressing or adenovirus-332 infected human epithelial cells. 333


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Ternary complexes formed by E4-ORF1 proteins from human adenovirus 334 subgroups A to D relocalize Dlg1 and PI3K to the plasma membrane. With respect 335 to PI3K activation, a key consequence of ternary complex formation by Ad9 E4-ORF1 is 336 relocalization of cytoplasmic Dlg1 and PI3K to the plasma membrane (17). To 337 determine whether ternary complexes formed by E4-ORF1 proteins from subgroups A 338 to C share this activity, we performed indirect immunofluorescence (IF) assays followed 339 by confocal microscopy on both HA-E4-ORF1 and wt E4-ORF1 lines double-labeled 340 with Dlg1 and p85α/β antibodies. In vector cells, Dlg1 and p85 were dispersed in the 341 cytoplasm, with some Dlg1 additionally detected at the plasma membrane (Fig. 6). 342 Compared to vector cells, the HA-E4-ORF1 and wt E4-ORF1 lines displayed increased 343 Dlg1 staining at the plasma membrane, where p85 also accumulated and co-localized 344 with Dlg1 in a high percentage of cells (Fig. 6). Whereas p85 was detected at the 345 plasma membrane in 0.84% of vector cells (n = 148 cells), the detection of p85 at the 346 plasma membrane of HA-E4-ORF1 lines or wt E4-ORF1 lines ranged from 79% to 90% 347 of cells (n ≥ 135 cells/cell line) or 83% to 94% of cells (n ≥ 185 cells/cell line), 348 respectively. We conclude that ternary complexes formed by E4-ORF1 proteins from 349 subgroups A to D promote translocation of Dlg1 and PI3K to the plasma membrane. 350 The E4-ORF1 protein from subgroup D Ad36 also forms the ternary complex that 351 mediates oncogenic Dlg1-dependent PI3K activation. E4-ORF1 has been reported 352 to be the primary determinant for obesity induced by Ad36 in infected experimental 353 animals and possibly also in people (24). This function depends on the ability of 354 Ad36 E4-ORF1 to activate PI3K, which modulates glucose and lipid metabolism in 355 adipose, skeletal muscle, and liver cells (25). Amino-acid sequences of the 125-residue 356


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E4-ORF1 proteins of Ad9 and Ad36 are closely related, displaying 92% identity and 357 differing only at only 10 residues. To determine whether the Ad36 E4-ORF1 protein 358 shares properties of other E4-ORF1 proteins investigated above, we generated 359 MCF10A lines stably transduced by pBABE encoding either wt or HA-tagged Ad36 360 E4-ORF1 (36ORF1 or HA-36ORF1 cells) and analyzed the two lines in key experiments. 361 Consistent with the high sequence identity between Ad9 and Ad36 E4-ORF1 proteins, 362 Ad9 E4-ORF1 antibody cross-reacted with Ad36 E4-ORF1 in immunoblots (Fig. 7A and 363 8A, lane 2). Like Ad9 E4-ORF1, wt and HA-tagged Ad36 E4-ORF1 migrated at 14-kDa 364 and ~20-kDa, respectively, in protein gels (data not shown). Extending our findings with 365 other E4-ORF1 proteins, Ad36 E4-ORF1 activated Akt in PI3K- and Dlg1-dependent 366 manners (Fig. 7A and B, compare lanes 1-2; Fig. 7C, compare lanes 3-4) but not 367 Erk1/2 (Fig. 7B, compare lanes 1 and 3). In addition, Ad36 E4-ORF1 elevated PI3K 368 protein levels (Fig. 7A and 8A and 8B, compare lanes 1-2), formed the ternary complex 369 (Fig. 8A, compare lanes 3-4; Fig. 8B, compare lanes 3-4 and lanes 5-6), and promoted 370 PI3K-dependent oncogenic cellular transformation of MCF10A cells (Fig. 8C). 371


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DISCUSSION 372 Constitutive activation of cellular PI3K is a conserved activity of E4-ORF1 from 373

subgroup A to D adenoviruses. This activity is crucial for several different E4-ORF1 374 functions, including oncogenic cellular transformation by Ad9, augmentation of viral 375 protein synthesis, replication and cell survival by Ad5, and dysregulation of cellular 376 glucose and lipid metabolism by Ad36. We recently demonstrated that PI3K activation 377 induced by subgroup D Ad9 E4-ORF1 depends on direct interactions with cellular Dlg1 378 and PI3K, resulting in formation of the Dlg1:E4-ORF1:PI3K ternary complex. In a 379 Dlg1-dependent fashion, this complex mediates upregulation of PI3K proteins and PI3K 380 signaling by recruiting activated PI3K to the plasma membrane (17). Prompted by 381 these findings, we asked whether E4-ORF1 from other adenoviruses shares this 382 molecular mechanism. The current study showed that stable expression of E4-ORF1 383 proteins from subgroups A to D in human MCF10A cells activates PI3K in a 384 Dlg1-dependent manner (Fig. 1 and 5A and 7A-C), elevates PI3K protein subunit levels 385 (Fig. 1B and 2A-B and 7A and 8B), and promotes ternary complex formation (Fig. 2 386 and 8A-B), Dlg1 and PI3K redistribution to the plasma membrane (Fig. 6), and 387 PI3K-dependent growth in soft agar (Fig. 3 and 8C). The former three E4-ORF1 388 activities were also demonstrated in cells infected with subgroup A to D adenoviruses 389 (Fig. 4 and 5B). Human adenovirus types 4 (Ad4) and 52 (Ad52), which individually 390 comprise adenovirus subgroup E or G, respectively, also encode an E4-ORF1 gene, 391 whereas human adenovirus types 40 and 41, comprising adenovirus subgroup F, do not. 392 It is not yet known whether the Ad4 and Ad52 E4-ORF1 proteins activate PI3K, though 393 they likely do based on their sequence similarity and shared conserved residues with 394


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E4-ORF1 proteins from subgroups A to D (unpublished data). We propose that most, if 395 not all, human adenovirus E4-ORF1 proteins use a conserved molecular mechanism to 396 dysregulate PI3K signaling in human epithelial cells. 397

Laprise et al. reported that Dlg1 is induced to bind directly to PI3K in a human 398 intestinal epithelial cell line and that the resulting Dlg1:PI3K complex activates PI3K 399 signaling to promote cellular differentiation (26). However, the current study showed 400 that Dlg1 associates with PI3K, via ternary complexes, in E4-ORF1-expressing cells but 401 not control MCF10A cells. It is unclear whether, unlike intestinal epithelial cells, 402 MCF10A cells do not form Dlg1:PI3K complexes under our experimental conditions or 403 whether our coIP assays are not sensitive enough to detect small quantities of the 404 complexes. Nonetheless, our results suggest that two separate E4-ORF1 activities act 405 in concert to produce significantly higher amounts of Dlg1:PI3K complexes in E4-ORF1-406 expressing MCF10A cells than in control MCF10A cells. We envision that E4-ORF1 407 first binds both Dlg1 and PI3K to tether them together constitutively within 408 Dlg1:E4-ORF1:PI3K ternary complexes. Next, by an undetermined mechanism, the 409 resulting ternary complexes upregulate PI3K protein levels by 10- to 20-fold in cells. 410 We postulate that this upregulation drives additional PI3K binding to E4-ORF1, resulting 411 in a positive feedback loop that amplifies ternary complex formation and abundance to 412 promote potent PI3K activation. 413

Ternary complex formation and PI3K activation by Ad9 E4-ORF1 require two 414 overlapping carboxyl-terminal E4-ORF1 protein interaction elements, the PDZ domain-415 binding motif (PBM) that mediates binding to Dlg1 and the KI residue pair required for 416 binding to both Dlg1 and PI3K (17). However, Ad9 E4-ORF1-induced PI3K activation 417


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additionally requires a third E4-ORF1 protein interaction element defined by seven 418 centrally-located residues designated as Domain 2 (27). A recent study reported that, in 419 Ad5-infected human epithelial cells, viral E4-ORF1 and E4-ORF6 form a heterocomplex 420 that binds and upregulates nuclear myc to activate transcription of myc-responsive 421 genes involved in anabolic glycolysis, reminiscent of the Warburg effect in cancer cells. 422 The activities of the heterocomplex increase nucleotide biosynthesis and augment viral 423 DNA synthesis and virion production (28). This study also showed that this E4-ORF1 424 activity does not depend on the Ad5 E4-ORF1 PBM that mediates binding to Dlg1, but 425 does depend on Ad5 E4-ORF1 residue D68, which is equivalent to Domain 2 426 residue D65 of Ad9 E4-ORF1 (27). These observations suggest the intriguing 427 possibility that Domain 2-dependent upregulation of myc-responsive glycolytic genes 428 acts in conjunction with the ternary complex to mediate E4-ORF1-induced PI3K 429 activation. A future study will test this hypothesis. 430

The first function identified for the human adenovirus E4-ORF1 gene was its role 431 as the primary oncogenic determinant for subgroup D Ad9-induced mammary 432 tumorigenesis in experimentally infected rats (4, 19, 29). Whereas the E1A and E1B 433 genes are dispensable for Ad9-induced tumorigenesis (30), they represent the major 434 oncogenic determinants for subgroup A and B adenoviruses that induce undifferentiated 435 sarcomas, and for subgroup C adenoviruses that are non-tumorigenic in experimentally 436 infected rodents (2). In addition, whereas E4-ORF1 from subgroup D Ad9 can promote 437 oncogenic transformation in rat cells, E4-ORF1 from subgroup A Ad12, subgroup B Ad3, 438 and subgroup C Ad5 cannot, which is caused, at least in part, by an expression defect 439 specific to E4-ORF1 proteins from subgroups A to C in rat cells (6). Contrary to findings 440


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in rat cells, the current study revealed that the four E4-ORF1 genes from subgroups A 441 to D, as well as subgroup D Ad36 E4-ORF1, can be stably expressed and similarly 442 induce the normal human MCF10A epithelial cell line to grow in soft agar (Fig 1A and 3), 443 an in vitro property correlating best with tumorigenic potential. These new findings 444 suggest that most, if not all, human adenovirus E4-ORF1 genes possess oncogenic 445 potential in human cells, raising potential safety concerns about current and future uses 446 of E4-ORF1-encoding vectors in human gene therapy and vaccination. 447

During the Ad5 life cycle, E4-ORF1-induced PI3K activation functions to enhance 448 late viral protein synthesis, S-phase progression of infected cells, and virion production 449 (9). Our results suggest that the ternary complex mediates these important E4-ORF1 450 functions. To assemble the ternary complex, the E4-ORF1 PBM must bind to 451 PDZ domains in cellular Dlg1 (7). As the PBM consists of a short peptide sequence 452 recognized by its cognate PDZ domain, PBM:PDZ domain interactions have been 453 proposed as promising targets for drug discovery (31). Small molecule inhibitors of the 454 E4-ORF1 PBM:Dlg1 PDZ domain interaction possibly could be designed to reduce 455 adenovirus replication in infected cells. Consequently, the conserved mechanism of 456 adenovirus-mediated PI3K activation revealed by our study may represent a useful 457 target for development of therapeutic drugs to treat and prevent adenovirus-associated 458 human diseases. 459


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ACKNOWLEDGMENTS 460 The work was supported by NIH grant R01CA58541 (RTJ) and by predoctoral 461 fellowship CPRIT #RP101499 (http://www.cprit.state.tx.us/funded-grants/) from Baylor 462 College of Medicine Comprehensive Cancer Training Program (KK). 463 464 We thank Susan Marriott for critiquing our manuscript, Nikhil Dhurandhar for the gift of 465 Ad36 DNA used to construct Ad36 E4-ORF1 plasmids, and reviewers whose comments 466 improved the manuscript. We also acknowledge the BCM Integrated Microscopy Core 467 facility for its contributions to confocal microscopy experiments. 468


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FIGURE LEGENDS 559 FIG 1. Human adenovirus E4-ORF1 proteins activate PI3K and elevate PI3K protein 560 levels. (A) Vector cells, rasV12 cells, and the indicated HA-E4-ORF1 lines were treated 561 with either DMSO vehicle (-) or 100 μM LY294002 (LY) (+) for 30 min. Cell extracts 562 were analyzed in immunoblot assays. (B) Extracts from vector cells and the indicated 563 wt E4-ORF1 lines were analyzed in immunoblot assays. 564 FIG 2. Human adenovirus E4-ORF1 proteins form ternary complexes. (A) Cell extracts 565 (300 μg of protein) from vector cells and the indicated HA-E4-ORF1 lines were IPed 566 with either p110α or Dlg1 antibody. Recovered proteins, as well as cell extract (input), 567 were analyzed in immunoblot assays. (B) Cell extracts from vector cells and the 568 indicated wt E4-ORF1 lines were IPed with Dlg1 antibody as described above in (A), 569 and recovered proteins, as well as cell extract (input), were analyzed in immunoblot 570 assays. 571 FIG 3. Human adenovirus E4-ORF1 proteins induce PI3K-dependent cellular 572 transformation. The indicated MCF10A lines treated with either DMSO vehicle (-) or 573 100 μM LY294002 (LY) (+) were analyzed in soft agar assays as described in the 574 Materials and Methods. LY treatment began on 2 days post plating. Cells were 575 photographed at 11 days post plating (40X magnification). 576 FIG 4. Human adenovirus E4-ORF1 proteins also form the ternary complex during viral 577 infections. (A) MCF10A cells were mock infected or infected with the indicated 578 wt adenovirus at a MOI of 1. Extracts (300 μg of protein) prepared at 24 hpi were IPed 579 with Dlg1 antibody. Recovered proteins, as well as cell extract (input), were analyzed in 580


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immunoblot assays. (B) MCF10A cells either mock infected or infected with the 581 indicated wt adenovirus at a MOI of 1 show either no cpe at 6 hpi or virus-specific cpe at 582 24 hpi. Cells were visualized by phase contrast microscopy (40X magnification). (C) 583 MCF10A cells either mock infected or infected with the indicated wt adenovirus at a MOI 584 of 1 accumulate either undetectable amounts or similar amounts of the major capsid 585 protein hexon at 6 hpi or 24 hpi, respectively. Cell extracts were resolved by 586 SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. 587 FIG 5. Human adenovirus E4-ORF1 proteins promote Dlg1-dependent PI3K activation 588 in both stable E4-ORF1-expressing and adenovirus-infected cells. (A) Extracts of 589 vector cells or the indicated HA-E4-ORF1 lines transduced with either the Dlg1 shRNA 590 (+) or the control scrambled shRNA (-) were analyzed in immunoblot assays. (B) Vector 591 cells transduced with either the Dlg1 shRNA (+) or the control scrambled shRNA (-) 592 were mock infected or infected with the indicated wt adenovirus at an MOI of 1. At 593 24 hpi, cell extracts were prepared and analyzed in immunoblot assays. 594 FIG 6. The ternary complex promotes translocation of cytoplasmic Dlg1 and PI3K to the 595 plasma membrane. Indirect immunofluorescence assays were performed with vector 596 cells and the indicated HA-E4-ORF1 and wt E4-ORF1 lines dually stained with p85α/β 597 (green) and Dlg1 (red) antibodies, followed by visualization by fluorescence confocal 598 microscopy, as described in the Materials and Methods. Nuclei were counterstained 599 with DAPI (blue). Individual and merged images are shown. White scale bar, 50 μm. 600 FIG 7. Ad36 E4-ORF1 is closely related to Ad9 E4-ORF1, activates PI3K in a 601 Dlg1-dependent manner, and elevates PI3K protein levels in MCF10A cells. (A) Ad36 602 E4-ORF1 elevates P-Akt and PI3K protein levels. Cell extracts from vector and 603


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36ORF1 cells were prepared and analyzed in immunoblot assays. (B) HA-tagged Ad36 604 E4-ORF1 activates Akt in a PI3K-dependent fashion but not Erk1/2 and elevates 605 PI3K proteins levels. Vector, HA-36ORF1, and rasV12 cells were treated with either 606 DMSO vehicle (-) or 100 μM LY294002 (LY) (+) for 30 min. Cell extracts were prepared 607 and analyzed in immunoblot assays. (C) HA-tagged Ad36 E4-ORF1 activates PI3K by 608 a Dlg1-dependent mechanism. Extracts of vector and HA-36ORF1 cells stably 609 expressing either the Dlg1 shRNA (+) or the control scrambled shRNA (-) were 610 prepared and analyzed in immunoblot assays. 611 FIG 8. Ad36 E4-ORF1 forms the ternary complex and induces MCF10A cell growth in 612 soft agar. (A) The wt Ad36 E4-ORF1 protein forms the ternary complex in MCF10A 613 cells. Cell extracts from vector and 36ORF1 cells were IPed with Dlg1 antibody as 614 described in Fig. 2, and recovered proteins, as well as cell extract (input), were 615 analyzed in immunoblot assays. (B) The HA-tagged Ad36 E4-ORF1 protein forms the 616 ternary complex in MCF10A cells. Cell extracts from vector and HA-36ORF1 cells were 617 IPed with either p110α or Dlg1 antibody as described in (A). Recovered proteins, as 618 well as cell extract (input), were analyzed in immunoblot assays. (C) Wt and HA-tagged 619 Ad36 E4-ORF1 proteins induce MCF10A cells to grow in soft agar. Vector, 36ORF1, 620 and HA-36ORF1 cells treated with either DMSO vehicle (-) or 100 μM LY294002 (LY) 621 (+) were analyzed in soft agar assays as described in the Materials and Methods. LY 622 treatment began on 2 days post plating. Cells were photographed at 11 days post 623 plating (40X magnification). 624 625


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TABLE 1 Reductions in P-Akt and Dlg1 protein 626 levels caused by expression of the Dlg1 shRNA in 627 adenovirus-infected MCF10A cellsa 628 Avg fold reductionb ± SEM 629 Virus infection P-Akt S473 Dlg1 630 Ad12 -3.0 ± 0.52 -3.2 ± 0.66 631 Ad3 -3.4 ± 0.17 -3.7 ± 0.97 632 Ad5 -2.6 ± 0.14 -3.5 ± 0.50 633 Ad9 -3.2 ± 0.68 -3.4 ± 0.47 634 aData from two independent experiments 635 bNormalized to matched adenovirus-infected 636 MCF10A cells expressing the negative-control 637 scrambled shRNA. 638 639


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