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

Date Published: May 1, 2014

Publisher: Public Library of Science

Author(s): Kathleen Kong, Manish Kumar, Midori Taruishi, Ronald T. Javier, Michael J. Imperiale.


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.

Partial Text

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 estrogen-dependent mammary tumors, and the E4-ORF1 gene is the primary viral oncogenic determinant [2]–[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].

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]–[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.




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