Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription

Abstract

Inhibition of p53-activated transcription is an integral part of the mechanism by which early region 1B 55K oncoprotein (E1B-55K) from adenovirus type 5 (Ad5) contributes to complete cell transformation in combination with Ad E1A. In addition, more recent data suggest that the mode of action of the Ad protein during transformation may involve additional functions and other protein interactions. In the present study, we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence, including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino-acid substitutions (C454S/C456S) created a 55K mutant protein, which had substantially reduced transforming activity. Interestingly, the same mutations neither affected binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA doublestrand break repair complex, and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.

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

“…Thus, it seems conclusive that adenoviruses evolved an E4orf3-dependent conserved mechanism to counteract this special PML-NB function (Stracker et al, 2005;Ullman et al, 2007;Ullman and Hearing, 2008). Intriguingly, our data provide evidence of E1B-55K as an additional viral factor interacting with and/or modulating cellular PML bodies as well as associated proteins (Sieber and Dobner, 2007;Ha¨rtl et al, 2008). Furthermore, these results in general offer a new perspective to deconstruct the complexity of Figure 4 Subnuclear colocalization of E1B-55K with endogenous PML during infection is dependent on a functional SCM motif.…”

“…Mutational inactivation of the NES leads to the accumulation of the viral protein in the nucleus accompanied by sequestration of different cellular proteins including PML (Endter et al, 2001(Endter et al, , 2005Ha¨rtl et al, 2008). To elucidate this phenomenon, stably transformed rat cell lines AB120/AB115 expressing E1A plus E1B-55K-wt/E1A plus E1B-55K-NES were analysed for steady-state localization of E1B-55K and PML (Figures 1A and B).…”

“…Subsequent steps of direct interaction (Sarnow et al, 1982a;Kao et al, 1990), transcriptional repression (Yew et al, 1994;Berk, 1998, 1999) and nuclear-cytoplasmic relocalization (Endter et al, 2001(Endter et al, , 2005 induce the complete silencing of p53-dependent tumour suppressive functions. However, previously described results suggest additional p53-independent mechanisms of E1B-55K induced cellular transformation involving cellular factors as Mre11 (Ha¨rtl et al, 2008) or the transcription factor Daxx (Sieber and Dobner, 2007). Interestingly, most so far known interaction partners of E1B-55K are transient components of the nuclear promyelocytic leukaemia (PML) bodies (Van Damme et al, 2010).…”

Distinct Requirements of Adenovirus E1b55K Protein for Degradation of Cellular Substrates

“…Many cellular proteins that interact with E1b55K localize at the aggresome in transfected and transformed cells (4,9,20,22,42,44,74). Therefore, protein aggregation has been proposed previously as a potential strategy utilized by E1b55K to inactivate cellular proteins (42) and promote transformation (31).…”

“…Export-deficient mutant forms of E1b55K accumulate in subnuclear aggregates that also contain cellular binding partners (20,31). In the absence of E4orf6, the expression of E1b55K produces cytoplasmic aggregates (17,27,37,42,47).…”