Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal.
A monoclonal antibody, 5H4, that recognizes human herpesvirus 7 (HHV-7) was used in Western analysis to probe HHV-7-infected SupT1 cells. This antibody recognizes a 40-kDa virus-specific polypeptide that is expressed in the absence of viral DNA synthesis. By screening a lambdagt11 HHV-7 cDNA library, the gene encoding the protein was identified as the U27 open reading frame previously reported [J. Virol. (1996) 70, 5975-5989]. Immunofluorescent studies showed a punctate nuclear localization of the protein in both HHV-7-infected cells and transfected cells. A computer program predicted two classic nuclear localization signals (NLSs) in the middle and C-terminal regions of the protein. A C-terminal deletion mutant of the protein could not enter the nucleus, whereas green fluorescent protein or maltose binding protein fused to the C-terminal region of the protein was transported into the nucleus. These findings demonstrate that the predicted C-terminal, but not middle, NLS of the protein actually function as NLS. In addition, nuclear transport of a maltose binding protein-fusion protein containing the C-terminal NLS of the U27 protein was inhibited by both wheat germ agglutinin and a Q69L Ran-GTP mutant, indicating that the U27 protein is transported into the nucleus from the cytoplasm by means of classic nuclear transport machinery. Interestingly, this NLS motif is highly conserved at the C-termini of all herpesvirus DNA polymerase processivity factors that have been examined.
Takeda
K
K
Department of Microbiology, Department of Anatomy and Cell Biology, Osaka University Medical School, 2-2 Yamada-oka, Osaka, Suita, 565-0871, Japan.
Haque
M
M
Nagoshi
E
E
Takemoto
M
M
Shimamoto
T
T
Yoneda
Y
Y
Yamanishi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D016199:Herpesvirus 7, Human / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D010449:Peptide Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000502:physiology
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D019913:Nuclear Localization Signals; D014764:Viral Proteins; D014909:Wheat Germ Agglutinins; D020931:ran GTP-Binding Protein