Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

Abstract

Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4 ؊ ) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4 ؊ virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCENuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication, and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5-encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a nonstructural subunit that is responsible for the gating functions of the NPC, and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

“…All the buffers were supplemented with one Complete Mini protease inhibitor cocktail tablet (Roche) per 10 ml. 32 P metabolic labeling was performed on apoptin as previously described (30). Following detection of the 32 P signal on film, the same gel was rehydrated and then stained with Coomassie to demonstrate equal expression of apoptin.…”

Regulation of Human Adenovirus Alternative RNA Splicing by the Adenoviral L4-33K and L4-22K Proteins

“…This inhibition is relieved by the viral E4-ORF4 protein, which induces SR protein dephosphorylation [ 26 , 27 ]. E4-ORF4 is a multifunctional viral regulator that binds to the cellular protein phosphatase PP2A and blocks E1A-induced transcription activation [ 28 , 29 , 30 ], induces hypo-phosphorylation of various viral and cellular proteins [ 28 , 31 ] and regulates adenovirus alternative RNA splicing [ 26 , 27 ]. The E4-ORF4 induced SR protein dephosphorylation relieves the repressive function of SR proteins on IIIa splicing and makes the branch point accessible for U2 snRNP recruitment [ 26 ].…”

“…Interestingly, DNA-PK is targeted by three additional adenoviral proteins, the E4-ORF3, E4-ORF4 and E4-ORF6 proteins [ 31 , 87 , 88 ]. Since DNA-PK is an essential factor involved in the double strand DNA repair pathway [ 89 ], the E4-ORF3 and E4-ORF6 proteins serve an important function to prevent concatemerization of the linear viral DNA genome during a lytic infection [ 88 , 90 , 91 ].…”

The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway

“…To date, several E4orf4 interacting partners besides PP2A have been reported and, in some cases, suggested to contribute to E4orf4-induced cell death, including c-Src (12,18,31,(70)(71)(72)(73)(74), the ATP-dependent chromatin-remodeling factor ACF (3), the NTPDASE4 gene product Golgi UDPase (20,29), and Nup 205 (2). In an attempt to discover more E4orf4 binding partners, we performed affinity purification/mass spectrometry (AP-MS) using both wild-type E4orf4 and class I and class II E4orf4 mutants.…”

“…uring infection by human adenovirus, the E4orf4 protein product is believed to enhance replication at least in part by introducing novel substrates to protein phosphatase 2A (PP2A) through interactions with one of its major classes of binding partners, the B55 family of PP2A regulatory subunits (1)(2)(3)(4)(5)(6). However, when expressed alone at high levels, E4orf4 induces the selective p53-independent death of a variety of human tumor cells (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) and is toxic in the yeast Saccharomyces cerevisiae (22)(23)(24)(25)(26)(27)(28)(29).…”