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The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

Kong K, Kumar M, Taruishi M, Javier RT - PLoS Pathog. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
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.

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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 µm.
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ppat-1004102-g008: 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 µm.

Mentions: 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 PBM-dependent 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 cell-cell 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.


The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

Kong K, Kumar M, Taruishi M, Javier RT - PLoS Pathog. (2014)

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 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4006922&req=5

ppat-1004102-g008: 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 µm.
Mentions: 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 PBM-dependent 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 cell-cell 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus