Dataset Information

Localization and importance of the adenovirus E4orf4 protein during lytic infection.

ABSTRACT: The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells (C. O'Shea, et al., EMBO J. 24:1211-1221, 2005). Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.

SUBMITTER: Miron MJ

PROVIDER: S-EPMC2643762 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Localization and importance of the adenovirus E4orf4 protein during lytic infection.

Miron Marie-Joëlle MJ Blanchette Paola P Groitl Peter P Dallaire Frederic F Teodoro Jose G JG Li Suiyang S Dobner Thomas T Branton Philip E PE

Journal of virology 20081210 4

The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for vi ...[more]

PMID: 19073741

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Project description:The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.

Project description:BackgroundThe adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions.ResultsThe E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit.ConclusionThe E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it.SignificanceA novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.

Dataset Information

Localization and importance of the adenovirus E4orf4 protein during lytic infection.

Publications

Localization and importance of the adenovirus E4orf4 protein during lytic infection.

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