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.

Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7

“…Consistent with previous reports, the virus mutant Hpm4166 (lacking E4orf4) exhibited modestly higher E4orf6 protein expression (Fig. 2A, lane 4) (47) and therefore resulted in more efficient coimmunoprecipitation with endogenous HoxB7 (Fig. 2A, upper panel, lane 4).…”

Nucleolar proteomics and viral infection

“…It has been known for some time that adenovirus infection has a profound effect on the processing and export of rRNA as well as affecting nucleolar antigens [83]. More recently, we and others have reported on adenovirus proteins that are directed to the nucleolus and on the sequestration of nucleolar antigens B23.1, B23.2, nucleolin and UBF into viral replication centres [47,48,[84][85][86][87][88][89][90][91][92]. Most notably, we showed that UBF was functionally recruited into viral DNA replication centres apparently without affecting the function of RNA pol I.…”

Control the Host Cell Cycle: Viral Regulation of the Anaphase-Promoting Complex

“…However, it has not yet been shown that this regulation of the APC occurs during adenovirus infection. E4orf4 is nonessential for virus replication in cultured human cancer or primary cells, so how this protein impacts adenovirus biology remains elusive (41). Determining whether E4orf4 inhibits the APC during infection, elucidating its consequence on virus replication, and identifying its mechanism of action are key questions to address in future studies.…”