Demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy. Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus, influenza A virus, avian coronavirus infectious bronchitis virus or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins. Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal overlapping with their nuclear localisation signal, which governs their nucleolar localisation. AbMole BioScience Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation. Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions. While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive. The CBDs are typically characterized by an eight stranded jellyroll b-sandwich, flanked by helices at the N- and C-termini as well as a small intervening helix situated between strands b6 and b7. Recent methods aimed at comparing patterns of amino acid conservations in sequence and in space have identified four conserved structural elements that are universally present in eukaryotic CBDs: the N-terminal helical bundle, the b2- b3 loop, the phosphate binding cassette and the hinge helix. Previous investigations on the CBD of EPAC1, have established the former three structural elements as crucial determinants underlying auto-inhibition. However, the role of the hinge helix as an auto-inhibitory determinant of the EPAC CBD is currently not fully understood. The last two turns of the EPAC hinge helix partially unfold as a6 rotates towards the a5 helix of the PBC upon cAMP binding. This hinge rotation has been rationalized as a consequence of the cAMP-induced repositioning of the PBC L273 residue, which contacts with F300 in the hinge helix. The repositioning of the conserved L273, and consequently F300, retracts the hinge helix toward the PBC helix upon activation. Recent studies mapping the EPAC allosteric network through chemical shift covariance analysis have revealed that L273 and F300 are part of a larger cluster of allosteric residues, which includes also a hydrophobic spine at the interface between the a4 and a6 helices. Such spine spans residues in the C-terminal end of the hinge helix that unwinds upon cAMP binding. Based on these observations, here we hypothesize that the Cterminal residues of the hinge helix are key determinants of EPAC auto-inhibition and that perturbations that destabilize the helix or induce unwinding shift the apo/inactive vs. apo/active pre-equilibrium toward the latter state, i.e. an active state without cAMP. To test this hypothesis, we designed three successive deletion mutations of the 149–318 EPAC1 construct, which spans the CBD and which from here on forth will be referred to as the Wt-EPAC. Specifically, these mutants are C-terminally truncated at positions 305, 310, and 312 and act as perturbations that destabilize the hinge helix of apoEPAC, mimicking the cAMP-induced unwinding.