The potential terminase subunit of human cytomegalovirus, pUL56, is translocated into the nucleus by its own nuclear localization signal and interacts with importin alpha.
Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be involved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence analysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucleus of infected cells. Solitary expression of wild-type as well as epitope-tagged pUL56 also resulted in nuclear distribution after transfection, suggesting the presence of an endogenous nuclear localization signal (NLS). Deletion of a carboxy-terminal stretch of basic amino acids (aa 816-827) prevented nuclear translocation, indicating that the sequence RRVRATRKRPRR of HCMV pUL56 mediates nuclear targetting. The signal character of the NLS sequence was demonstrated by successful transfer of the NLS to a reporter protein chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions within the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation. In order to identify the transport machinery involved, the potential of pUL56 to bind importin alpha (hSRP1 alpha) was examined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1 alpha was provided by in vitro binding assays. In view of these findings, it is suggested that nuclear translocation of HCMV pUL56 is mediated by the importin-dependent pathway.
Giesen
Kyra
K
Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany1.
Radsak
Klaus
K
Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany1.
Bogner
Elke
E
Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany1.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D003587:Cytomegalovirus / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D028884:Karyopherins; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000737:chemistry / Q000378:metabolism*
D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C090238:UL56 protein, cytomegalovirus; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004706:Endodeoxyribonucleases; C030894:terminase