Nuclear-cytoplasmic shuttling is not required for the Epstein-Barr virus EBNA-LP transcriptional coactivation function.

Epstein-Barr virus (EBV) EBNA-LP is a transcriptional coactivator of EBNA2 that works though interaction with the promyelocytic leukemia nuclear-body-associated protein Sp100A. EBNA-LP localizes predominantly in the nucleus through the action of nuclear localization signals in the repeated regions of the protein. EBNA-LP has also been detected in the cytoplasm, and a previous study suggested that some of the EBNA-LP coactivation function is mediated by relocalizing histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. Although EBNA-LP can be found in the cytoplasm, it has no obvious nuclear export signal, and there is no direct evidence for active shuttling between these cellular compartments. Whether active shuttling between the nucleus and cytoplasm is required for coactivation remains to be clarified. To address these issues, we tested a variety of EBNA-LP isoforms and mutants for nuclear-cytoplasmic shuttling activity in an interspecies heterokaryon assay and for the ability to associate with HDAC4. EBNA-LP isoforms smaller than 42 kDa shuttle efficiently in the heterokaryon assay via a crm-1-independent mechanism. In addition, no specific EBNA-LP domain that mediates nuclear export could be identified. In contrast, an EBNA-LP 62-kDa isoform does not demonstrate detectable shuttling in the heterokaryon assay yet still coactivates EBNA2 similarly to the smaller EBNA-LP isoforms. All of the EBNA-LP mutants tested, including the coactivation-deficient DeltaCR3 mutant and the nonshuttling 62-kDa isoform, were capable of associating with HDAC4. Taken together, our results suggest that simple diffusion may account for the nuclear export observed with smaller isoforms of EBNA-LP, that nuclear-cytoplasmic shuttling is not required for efficient EBNA-LP coactivation function, and that competence for HDAC4 association is not sufficient to mediate nuclear-cytoplasmic shuttling or EBNA-LP coactivation in the absence of a functional interaction with Sp100A.