title: Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-beta-galactosidase fusion proteins.
abstract: The 605 amino acid E1 protein of bovine papillomavirus type 1 (BPV-1) is a multifunctional nuclear protein required for viral DNA replication. A nuclear localization signal (NLS) sequence was previously defined by point mutations in three short adjacent clusters of basic amino acids located in the amino-terminal region of the E1 protein. In this study, we used a fusion protein approach to evaluate the contribution of other regions of the E1 protein to nuclear transport. The nearly full-length E1 gene and six non-overlapping subfragments were each fused in-frame with the lacZ gene in a eukaryotic expression vector. Each clone was electroporated into COS-1 cells, and the intracellular location of the E1-beta-galactosidase fusion proteins was determined by immunofluorescence. Only the constructs containing the full-length E1 or a single subregion (E1-259; amino acids 84 to 166) produced fusion proteins that entered the nucleus. Point mutations in the NLS sequences of the E1-259-lacZ construct prevented nuclear translocation of the corresponding fusion protein. This confirms the previous result that the cluster of basic amino acids is critical for nuclear transport. Furthermore, the data obtained in this investigation indicated that the region of E1 containing the NLS sequence was not only necessary, but was also sufficient for nuclear localization. No other region of E1 contained independent nuclear localization activity.
authors.affiliation: Department of Medical Microbiology and Immunology, Texas A & M University Health Science Center, College Station 77843-1114.
authors.forename: X
authors.initials: X
authors.lastname: Leng
authors.forename: V G
authors.initials: VG
authors.lastname: Wilson
authors.forename: X L
authors.initials: XL
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D052688:Bovine papillomavirus 1 / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis*; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
chemical_list: D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D001616:beta-Galactosidase