Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50

The murine cytomegalovirus (MCMV) M44 gene product pp50 is normally present in the nuclei of virus-infected cells. During transient expression of pp50 in COS-1 cells, the phosphoprotein was readily detectable in the nuclei, i ndicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 su ggested that alterations in both the C terminus and the highly conserved N- terminal domains of pp50 affect nuclear localization. In particular, the C- terminal 11 amino acids of pp50, which includes a "KKQK" motif, were able t o mediate the import of a beta-galactosidase fusion protein into the nucleu s. The pair of lysine residues in this motif constitutes an essential eleme nt: of the C-terminal NLs as mutation of this motif to AAQK directly affect ed the nuclear localization of either pp50 or beta-galactosidase fusion pro teins containing the C-terminal portion of pp50. Furthermore our results in dicated that the functionality of the C-terminal NLS is dependent on the st ructural integrity of the highly conserved N-terminal portion of the molecu le, as deletion of amino acids 157-201 alone adversely affected nuclear loc alization. In the absence of a functional C-terminal NLS, the subcellular l ocalization of pp50 is sensitive to potential conformational changes induce d by mutations within the N-terminal half of the molecule. Under those circ umstances, mutation of the YK residues at position 22-23 or deletion of ami no acids 267-283 was sufficient to produce a protein that was impaired in n uclear import or retention. (C) 1999 Academic Press.