A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway.
The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.
Lischka
Peter
P
Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany.
Sorg
Gabriele
G
Kann
Michael
M
Winkler
Michael
M
Stamminger
Thomas
T
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000737:chemistry / Q000378:metabolism*; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism*
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C072218:UL84 protein, Cytomegalovirus