JOURNAL OF VIROLOGY, Aug. 2002, p. 8460–8467 Vol. 76, No. 16 0022-538X/02/$04.00ϩ0 DOI: 10.1128/JVI.76.16.8460–8467.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Characterization of the Nuclear Localization Signal of the Borna Disease Virus Polymerase Michelle Portlance Walker† and W. Ian Lipkin* Emerging Diseases Laboratory, Departments of Neurology, Anatomy, and Neurobiology and Microbiology and Molecular Genetics, University of California—Irvine, Irvine, California 92697-4292 Received 21 December 2001/Accepted 30 April 2002 Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. BDV proteins involved in replication and transcription must pass through the nuclear envelope to associate with the genomic viral RNA. The RNA-dependent RNA polymerase (L) of BDV is postulated to be the catalytic enzyme of replication and transcription. We demonstrated previously that BDV L localizes to the nucleus of BDV-infected cells and L-transfected cells. Nuclear local- Downloaded from ization of the protein presupposes the presence of a nuclear localization signal (NLS) within its primary amino acid sequence or cotransport to the nucleus with another karyophilic protein. Because L localized to the nucleus in the absence of other viral proteins, we investigated the possibility that L contains an NLS. The minimal sequence required for nuclear localization of L was identified by analyzing the subcellular distribution of deletion mutants of L fused to a flag epitope tag or ␤-galactosidase. Although the majority of the L fusion proteins localized to the cytoplasm of transfected BSR-T7 cells, a strong NLS (844RVVKLRIAP852) with basic jvi.asm.org and proline residues was identified. Mutation of this sequence resulted in cytoplasmic distribution of L, confirming that this sequence was necessary and sufficient to drive the nuclear localization of L. Borna disease virus (BDV) transcribes and replicates its cleoprotein (N) (8, 10), phosphoprotein (P) (12, 13), and X at COLUMBIA UNIVERSITY on May 21, 2010 nonsegmented negative-strand (NNS) RNA genome in the protein (X) (17). The N-NLS is similar in sequence and amino- nucleus of infected cells. This nuclear phase of the virus life terminal position to the NLSs of the VP1 proteins of simian cycle is unique among members of the NNS RNA animal virus 40 (SV40) and polyomavirus (10). In contrast, P contains viruses (3, 5). Following translation in the cytoplasm, BDV two NLSs (12, 13). The first is located near the amino-terminal proteins must travel through the nuclear membrane and into portion of P and is bipartite, similar to the nucleoplasmin NLS the nucleus in order to participate in virus transcription. Three (12). The second is located at the carboxyl-terminal portion of mechanisms are described to account for cytoplasmic-nuclear P (12, 13); both P-NLSs are unique in that proline residues trafficking: passive diffusion, active transport, and cotransport play a central role in their activity (13). X import is mediated (16). by interaction of a nonconventional karyophilic signal at its Active transport requires soluble cytoplasmic receptors amino terminus with importin-␣ (17). called karyopherins (in yeast cells) or importins (in mamma- We demonstrated previously (15) that BDV P and the lian cells), energy in the form of GTP (in many but not all RNA-dependent RNA polymerase (L) interact. This inter- cases), and a specific nuclear localization signal(s) (NLS[s]) action alone could lead to nuclear localization of L due to within the primary amino acid sequence of the karyophilic the P-NLS; however, immunohistochemical studies with an- protein (6). At least four active transport pathways have been ti-L1 antisera and BSR-T7 cells (Huh-7 cells stably trans- described previously (1). Each requires that importins recog- fected to express T7 RNA polymerase) transiently trans- nize and interact directly with the NLS of the protein to be fected with an L-expression plasmid revealed the presence imported. The importin-NLS protein complex then docks at of L protein in the nucleus (15). This finding suggested the fibrils extending from the nuclear pore complex and is im- presence of an NLS(s) in L. To characterize the putative ported into the nucleus through a series of interactions that L-NLS, immunofluorescence analyses of BSR-T7 cells trans- utilize energy in the form of GTP. Cotransport occurs when a fected with wild-type or mutant forms of L fused to a flag protein that lacks an NLS and is too large for passive diffusion epitope tag or ␤-galactosidase were performed. Analysis of interacts with a protein that contains a functional NLS (1). The proteins are subsequently cotransported into the nucleus via amino- and carboxyl-truncation mutants fused to the flag active transport due to the presence of the NLS. epitope tag indicated that the central residues of L (amino Functional NLSs have been mapped within the BDV nu- acids 824 to 1062) were sufficient for nuclear localization. These results were confirmed and expanded by analysis of L-␤-galactosidase fusion constructs. A strong NLS at resi- * Corresponding author. Mailing address: Center for Immuno- dues 844 to 852 was identified. Mutation of 844R (arginine) pathogenesis and Infectious Diseases, Mailman School of Public and 847K (lysine) to A (alanine) led to cytoplasmic accu- Health, Columbia University, 722 W. 168th St., New York, NY 10032. mulation of L, confirming that these residues within the Phone: (212) 305-0695. Fax: (212) 305-9413. E-mail: wil@columbia .edu. sequence 844RVVKLRIAP852 are necessary and sufficient † Drug Discovery, Ribapharm, Inc., Costa Mesa, CA 92626. for nuclear localization of L. 8460 VOL. 76, 2002 NOTES 8461 Downloaded from jvi.asm.org at COLUMBIA UNIVERSITY on May 21, 2010 FIG. 1. Subcellular localization of L-flag epitope tag fusion proteins. (A) Description of expression plasmids and summary of results. Amino acid sequence of L, with a diagram of full-length and deletion mutant L-flag epitope tag fusion expression plasmids. Plasmid names and subcellular localization of fusion proteins are listed. (B through G) Subcellular localization of full-length or deletion mutants of L-flag epitope tag by indirect immunofluorescence in BSR-T7 cells transfected with expression plasmids. (B) pTM-TL; (C) pTM-T⌬C759; (D) pTM-T⌬C180; (E) pTM- T⌬N1062; (F) pTM-T⌬N394; (G) pTM1. Cells were stained with anti-flag M2 murine antibody (Sigma) and goat anti-mouse IgG fluorescein isothiocyanate (Caltag). 8462 NOTES J. VIROL. Downloaded from FIG. 2. LacZ-L fusion plasmid description and subcellular distribution of full-length or deletion mutants of ␤-galactosidase-L by indirect immunofluorescence in BSR-T7 cells transfected with expression plasmids. (A) Amino acid sequence of L, with full-length and deletion mutant jvi.asm.org LacZ-L fusion constructs. Plasmid names and subcellular localization of fusion proteins are listed. (B) Cells were stained with murine anti-␤- galactosidase antibody (Sigma) and anti-mouse IgG fluorescein isothiocyanate (secondary antibody) (Caltag). (a) pTM-ZL; (b) pTM-Z⌬C824; (c) pTM-Z⌬C759; (d) pTM-Z⌬C180; (e) pTM-Z⌬N1439; (f) pTM-Z⌬N1062; (g) pTM-Z⌬N824; (h) pTM-Z⌬N561; (i) pTM-LacZ; (j) pTM1 (empty vector, negative control). at COLUMBIA UNIVERSITY on May 21, 2010 Subcellular distribution of L-flag epitope tag fusions. The Z⌬C180) (Fig. 22B, panel d) aa from the carboxyl region of L subcellular distribution of L-flag epitope tag fusion proteins led to cytoplasmic staining. In contrast, deletion of 561 (pTM- was determined immunohistochemically with anti-flag M2 mu- Z⌬N561) (Fig. 2B, panel h) or 824 (pTM-Z⌬N824) (Fig. 2B, rine antibody (Sigma) and goat anti-mouse immunoglobulin G panel g) aa from the amino region of L retained the nuclear (IgG) fluorescein isothiocyanate (Caltag) in BSR-T7 cells tran- staining pattern. Further deletion from the amino region of L siently transfected with expression plasmids encoding the flag again led to cytoplasmic localization of the fusion proteins, as epitope tag fused to amino- or carboxyl-deletion mutants of observed upon deletion of 1,062 (pTM-Z⌬N1062) (Fig. 2B, BDV L. Both full-length L fused to the flag epitope tag (pTM- panel f) or 1,439 (pTM-Z⌬N1439) (Fig. 2B, panel e) amino TL) and the 394-amino-acid (aa) amino-terminal deletion mu- residues. Fusion of L aa 824 to 1062 (pTM-Z⌬N824C1062) ⌬ tant (pTM-T N394) fused to the flag epitope tag localized to (Fig. 3C, panel a), 824 to 941 (pTM-Z⌬N824C941) (Fig. 3C, the nucleus of transfected BSR-T7 cells (Fig. 1A, B, and F). In panel e) or 824 to 853 (pTM-Z⌬N824C853) (Fig. 3C, panel f) contrast, the 953-aa carboxyl-terminal deletion mutant fused to led to nuclear localization of ␤-galactosidase. In contrast, all the flag epitope tag (pTM-T⌬C759) (Fig. 1A and C) and the ␤-galactosidase-L fusions that contained L sequence carboxyl 1,062-aa amino-terminal deletion mutant fused to the flag to aa 854 localized to the cytoplasm: aa 921 to 1062 (pTM- epitope tag (pTM-T⌬N1062) (Fig. 1A and E) localized to the Z⌬N921C1062) (Fig. 3C, panel b), 921 to 1015 (pTM- cytoplasm. Deletion of 1,532 aa from the carboxyl region of L Z⌬N921C1015) (Fig. 3C, panel c), or 854 to 1,015 (pTM- (pTM-Z⌬C180) resulted in predominantly cytoplasmic fusion Z⌬N854C1015) (Fig. 3C, panel d). No fluorescence was protein (Fig. 1A and D). No fluorescence was observed in cells observed in cells transfected with empty vector (pTM1) (Fig. transfected with vector (pTM1) alone (Fig. 1G). Subcellular localization of ␤-galactosidase-L fusion pro- 2B, panel j and 3C, panel g). teins. The subcellular distribution of ␤-galactosidase-L fusion L-NLS mutant localizes to the cytoplasm.