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, indicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 suggested 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 to mediate the import of a beta-galactosidase fusion protein into the nucleus. The pair of lysine residues in this motif constitutes an essential element of the C-terminal NLS as mutation of this motif to AAQK directly affected the nuclear localization of either pp50 or beta-galactosidase fusion proteins containing the C-terminal portion of pp50. Furthermore our results indicated that the functionality of the C-terminal NLS is dependent on the structural integrity of the highly conserved N-terminal portion of the molecule, as deletion of amino acids 157-201 alone adversely affected nuclear localization. In the absence of a functional C-terminal NLS, the subcellular localization of pp50 is sensitive to potential conformational changes induced by mutations within the N-terminal half of the molecule. Under those circumstances, mutation of the YK residues at position 22-23 or deletion of amino acids 267-283 was sufficient to produce a protein that was impaired in nuclear import or retention.
Loh
L C
LC
Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada. loh@sask.usask.ca
Keeler
V D
VD
Shanley
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002467:Cell Nucleus / Q000821:virology; D004268:DNA-Binding Proteins / Q000235:genetics*; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D018146:Muromegalovirus / Q000235:genetics*; D017421:Sequence Analysis; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000235:genetics*
D004268:DNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C087085:phosphoprotein 50, cytomegalovirus