Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.

Abstract

Abstract. The PML protein was first identified as part of a fusion product with the retinoic acid receptor (RARa), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RARa hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.T HE eukaryotic nucleus is highly organized into discrete domains which spatially separate different biochemical processes. A variety of metabolic activities such as DNA replication, ribosome assembly, transcription, and pre-mRNA processing localize to distinct subnuclear compartments. The most conspicuous example is the nucleolus in which rRNAs are assembled into ribosomal subunits (reviewed by Scheer and Weisenberger, 1994). DNA replication sites were also shown to concentrate within discrete regions containing the proliferating cell nuclear antigen (PCNA; 1 Bravo and MacdonaldBravo, 1987) as well as DNA methyltransferase (Leonhardt et al., 1992). In addition, the small nuclear ribonucleoprotein particles (snRNPs), which are the major subunits of spliceosomes, were similarly found to localize to T. Carvalho and J.-S. Seeler should be considered as first authors.

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…Representative results of IF analysis is shown in Figure 4A. As expected, PML is relocalized in virus-infected cells (Figures 4Ab, Ae, Ah, and Ak) by E4orf3 in so-called track-like structures (Carvalho et al, 1995;PuvionDutilleul et al, 1995;Doucas et al, 1996), whereas E1B-55K shows a more complex localization pattern (Ornelles and Shenk, 1991). However, a subpopulation of about 30% of infected HepaRG cells exhibit small nuclear accumulations of the viral protein (Figure 4Aa).…”

“…Over the last years, it has become evident that cellular PML bodies facilitate various functions in cell metabolism including cell differentiation (Salomoni, 2009) (Carvalho et al, 1995;Puvion-Dutilleul et al, 1995;Doucas et al, 1996). Furthermore, PML bodies are proposed to mediate a general intracellular barrier for viral infections (Everett, 2001;Everett and ChelbiAlix, 2007;Tavalai and Stamminger, 2008).…”

“…Viral infection alters the affinity of E1B-55K for specific PML isoforms Previously published data illustrate that the adenoviral protein E4orf3 is necessary and sufficient to disrupt cellular PML bodies during adenoviral infection (Carvalho et al, 1995;Puvion-Dutilleul et al, 1995;Doucas et al, 1996). As the E4orf3-mediated PML relocalization seems to be conserved among human adenoviruses (Stracker et al, 2005), it has been proposed that this might be a general mechanism to counteract the antiviral capabilities of PML bodies (Ullman et al, 2007;Ullman and Hearing, 2008).…”

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor1α promoter

“…However, extended use of the E4ORF3 gene product in the clinic is likely to be antigenic and may be complicated by the variety of effects it mediates in the cell, including the induction of changes in host cell nuclear structure. 23 Although the precise mechanism is unknown, the E4ORF3 protein may act directly or indirectly on the promoter 9,11 to increase persistence. Together these observations are consistent with promoter attenuation being responsible for transient gene expression in the mouse lung.…”

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2

“…Although, it was proposed that a complex of E4orf6 and E1B-55K mediates viral RNA transport in which E4orf6 confers nuclear import and export (Dobbelstein et al, 1997), the detailed mechanism by which these adenovirus proteins promote nucleo-cytoplasmic RNA transport is not completely understood. Recent studies indicate that E4orf6, and in particular E4orf3, initiate the nuclear reorganization of cellular and viral factors that might also indirectly favour this process (Carvalho et al, 1995;Doucas et al, 1996). At late times of infection E1B-55K and E4orf6 are localized within and at the periphery of nuclear viral inclusions, believed to be the sites of viral transcription and/or replication (Ornelles and Shenk, 1991).…”