Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication. The nuclear import of E1A is mediated by a nuclear localization signal; however, whether E1A can be actively exported from the nucleus is unknown. We first reported a CRM1-dependent nuclear export signal (NES) in E1A that is conserved in the group C adenoviruses. We showed that CRM1 and E1A coimmunoprecipitated and that blockage of CRM1 function by leptomycin B or small interfering RNA resulted in the nuclear localization of E1A. Through mutational analyses, we identified an active canonical NES element within the E1A protein spanning amino acids 70-80. We further demonstrated that phosphorylation of adjacent serine (S)89 resulted in the cytoplasmic accumulation of E1A. Interestingly, coincident with the accumulation of cells in the S/G₂/M phase and histone H1 phosphorylation, E1A was relocated to the cytoplasm at the late stage of the viral cycle, which was blocked by the CDC2/CDK2 inhibitor roscovitine. Importantly, titration of the progenies of the viruses in infected cells showed that the replication efficiency of the NES mutant adenovirus was up to 500-fold lower than that of the wild-type adenovirus. Collectively, our data demonstrate the existence of a NES in E1A that is modulated by the phosphorylation of the S89 residue and the NES plays a role for an efficient viral replication in the host cells.--Jiang, H., Olson, M. V., Medrano, D. R., Lee, O. H., Xu, J., Piao, Y., Alonso, M. M., Gomez-Manzano, C., Hung, M. C., Yung, W. K. A., Fueyo, J. A novel CRM1-dependent nuclear export signal in adenoviral E1A protein regulated by phosphorylation.