Human adenovirus type 5 E4orf4 interacts wih the nucleopore component nup205 to regulate viral gene expression and replication

The Adenovirus type 5 E4orf4 protein (E4orf4) is a multifunctional protein that regulates viral transcription and splicing. Much of the activity of E4orf4 is thought to be mediated through binding of the Protein Phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A resulting in dephosphorylation of host factors, such as SR splicing factors and the AP-1 transcription factor. However, the full complement of cellular factors that interact with E4orf4, and the molecular mechanisms that E4orf4 utilizes during replication to regulate gene expression remain poorly understood. In the following study, we utilized immunoprecipitation followed by mass spectrometry to identify novel interacting proteins with E4orf4. Interestingly, we identified a nucleoporin, Nup 205, a component of the nuclear pore complex as an interacting partner. We show that the Arginine Rich Motif (ARM) of E4orf4 is required for interaction with Nup 205 and for nuclear localization of E4orf4. ARMs are commonly found on many viral nuclear proteins. Interestingly, we observed that Nup 205 interacts with three different viral nuclear proteins containing ARMs. In each case, viral ARM proteins also bound to the same region on Nup 205. Point mutations of the ARM sequence on each of the three viral proteins resulted in loss of interaction with Nup 205 and loss of nuclear localization of the viral protein. Nup 205 may therefore represent a common cellular target for viruses encoding ARM containing proteins. We have further tested the role of E4orf4 and Nup 205 in adenovirus replication and gene expression. Previous studies have shown that compared to wild type adenovirus, E4orf4 deficient adenovirus (Orf4-) have elevated E1A and E4orf6 expressions and reduced late protein production. Such effects are phenotypically copied by the loss of Nup 205, where wild type adenovirus infecting H1299 cells with reduced level of Nup 205 also results in elevated E1A and E4orf6 and reduced late protein production. Furthermore, knockdown of Nup 205 resulted in reduced cytopathic effect and a more than four-fold reduction in the replication of wild type adenovirus. Taken together, these data suggest Nup 205 is required by adenovirus for proper regulation of gene expressions and viral replication, and that interaction with E4orf4 may mediate these effects. Since E4orf4 is known to deregulate phosphorylation of its target proteins, our future studies will focus on identifying phosphorylation sites on Nup 205 and determining the effect of E4orf4 on such sites and nucleopore structure and function.

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