Characterization of the Nuclear Localization Signal of Borna Disease

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.

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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. Analysis of L ␤ proteins was assessed by indirect immunofluorescence, using truncation mutants fused with the flag epitope tag and -ga- murine anti-␤-galactosidase (primary antibody) (Sigma) and lactosidase suggested that an L-NLS was located between aa anti-mouse IgG fluorescein isothiocyanate (secondary anti- 824 to 853. To test this hypothesis, BSR-T7 cells were trans- body) (Caltag) after transient transfection of BSR-T7 cells fected with a plasmid encoding L-NLS mutants Arg844Ala and with plasmids that encode ␤-galactosidase fused to amino- or Lys847Ala (R844A/K847A). Subcellular distribution of the carboxyl-deletion mutants of BDV L (Fig. 2A). Whereas ␤-ga- mutant protein within transfected cells was assessed by indirect lactosidase alone was cytoplasmic (Fig. 2B, panel i), ␤-galac- immunofluorescence using murine anti-L1 primary antisera tosidase fused to wild-type L (pTM-ZL) (Fig. 2B, panel a) was and anti-mouse IgG fluorescein isothiocyanate (secondary an- nuclear. Deletion of 888 (pTM-Z⌬C824) (Fig. 2B, panel b), tibody) (Caltag). Whereas wild-type L localized to the nucleus 953 (pTM-Z⌬C759) (Fig. 2B, panel c), or 1,532 (pTM- (Fig. 4b), the mutant protein localized to the cytoplasm (Fig. VOL. 76, 2002 NOTES 8463 Downloaded from jvi.asm.org at COLUMBIA UNIVERSITY on May 21, 2010

FIG. 2—Continued.

4a). No fluorescence was observed in cells transfected with (Fig. 7), followed by goat anti-mouse IgG secondary antibody vector (pTM1) alone (Fig. 4c). conjugated to horseradish peroxidase (Sigma). L-flag epitope- Expression of L-fusion proteins and mutant proteins of the tagged fusion proteins of the predicted sizes were detected as predicted size. The size of proteins expressed in BSR-T7 cells follows: pTM-T⌬C180, 20 kDa (Fig. 5A, lane 1); pTM- transfected with plasmids encoding ␤-galactosidase-L fusions, T⌬N1062, 85 kDa (Fig. 5B, lane 1); pTM-T⌬C759, 95 kDa L-flag epitope tag fusions, or the L-NLS mutant was deter- (Fig. 5B, lane 2); pTM-T⌬N394, 150 kDa (Fig. 5B, lane 3);and mined by Western immunoblotting using murine anti-flag M2 pTM-TL, 190 kDa (Fig. 5C, lane 2). Fusions of ␤-galactosi- primary antibody (Sigma) (Fig. 5), murine anti-␤-galactosidase dase-L expressed from plasmids were also consistent as far as primary antibody (Sigma) (Fig. 6), or murine anti-L1 antisera their predicted sizes: pTM-ZL, 310 kDa (Fig. 6A, lane 2); 8464 NOTES J. VIROL. Downloaded from jvi.asm.org at COLUMBIA UNIVERSITY on May 21, 2010

FIG. 3. B-galactosidase-L fusion constructs and subcellular distribution of deletion mutants of ␤-galactosidase-L by indirect immunofluorescence in BSR-T7 cells transiently transfected with expression plasmids. (A) Map of the L open reading frame (ORF) (nucleotides 2472 to 3269), indicating restriction endonuclease sites, PCR primers, and subcellular localization. (B) Primer names, sequences, and nucleotide positions within the L ORF. (C) Subcellular distribution of deletion mutants of ␤-galactosidase-L by indirect immunofluorescence in BSR-T7 cells transiently transfected with expression plasmids. Cells were stained with anti-␤-galactosidase murine antibody (Sigma) and anti-mouse IgG fluorescein isothiocyanate (secondary antibody) (Caltag). (a) pTM-Z⌬N824C1062; (b) pTM-Z⌬N921C1062; (c) pTM-Z⌬N921C1015; (d) pTM- Z⌬N854C1015; (e) pTM-Z⌬N824C941; (f) pTM-Z⌬N824C853 (g) pTM1 (empty vector, negative control).

pTM-Z⌬C824, 200 kDa (Fig. 6A, lane 3); pTM-Z⌬C759, 200 interacts with BDV-P (15), which has a strong NLS (12, 13); kDa (Fig. 6A, lane 4); pTM-T⌬C180, 140 kDa (Fig. 6A, lane thus, it is conceivable that BDV L is cotransported into the 5); pTM-Z⌬N1439, 150 kDa (Fig. 6A, lane 6); pTM-Z⌬N1062, nucleus with BDV P. However, in the absence of P, recombi- 190 kDa (Fig. 6A, lane 7); pTM-Z⌬N824, 220 kDa (Fig. 6A, lane nant L localizes to the nucleus of transfected BSR-T7 cells 8); pTM-Z⌬N561, 240 kDa (Fig. 6A, lane 9); pTM-Z⌬ (15), indicating the presence of an NLS, which we character- N824C1062, 146 kDa (Fig. 6B, lane 2); pTM-Z⌬N921C1062, 135 ized. kDa (Fig. 6B, lane 3); pTM-Z⌬N921C1015, 130 kDa (Fig. 6B, To identify the NLS of BDV L, a flag epitope tag was fused lane 4); pTM-Z⌬N854C1015, 140 kDa (Fig. 6B, lane 5); pTM- with the wild type and with amino and carboxyl deletions of L Z⌬N824C941, 133 kDa (Fig. 6B, lane 6); and pTM- (Fig. 1A). Indirect immunofluorescence with murine anti-flag Z⌬N824C853, 123 kDa (Fig. 6B, lane 7). L-NLS mutant protein M2 antibody (Sigma) on BSR-T7 cells transfected with expres- of the predicted size (190 kDa) was expressed from pTM-L- sion plasmids showed clear nuclear staining when residues 759 AVVA (Fig. 7, lane 2). Proteins consistent with the size of those to 1062 were present (Fig. 1B to F). Amino- and carboxyl- expressed from expression plasmids were not detected upon deletion mutants of L fused to ␤-galactosidase confirmed the transfection with empty vector (pTM-1) (Fig. 5A, lane 2; Fig. 5B, result observed with L-flag epitope tag fusions and were used lane 4; Fig. 5C, lane 1; Fig. 6, lanes 1; and Fig. 7, lane 1). to further narrow the sequence to residues 824 to 1062 (Fig. BDV replicates in the nucleus of infected cells (3, 5). Thus, 2B, panels a to i). the BDV polymerase must localize to the nucleus of infected Fine mapping of the L-NLS was performed by fusion of L to cells (15). Nuclear localization of BDV L could result from an ␤-galactosidase in order to ensure that fusion proteins were NLS or from cotransport with other viral proteins (16). BDV L more than 60 kDa, the threshold for passive diffusion into the VOL. 76, 2002 NOTES 8465 Downloaded from jvi.asm.org at COLUMBIA UNIVERSITY on May 21, 2010

FIG. 3—Continued.

nucleus (14). Ultimately, residues 824 to 853 were identified as localized to the cytoplasm of BSR-T7 cells (Fig. 4a). This sufficient to drive nuclear localization of the ␤-galactosidase region contained a stretch of basic residues similar to the SV40 fusion protein (Fig. 3C, panels a to f). class of NLSs (844RVVKLR849) (9). It is likely that these Analysis of this sequence indicated a strong NLS at residues residues represent the core L-NLS. Core NLSs are usually 844 to 854. The activity of the NLS was confirmed by mutating defined by a hexapeptide, with K and R residues and either V, the sequence within L (R844A/K847A). This L-NLS mutant P, or A residues interspersed (9). P, G, or acidic residues flank

FIG. 4. Subcellular localization of wild-type L and L-NLS mutant by indirect immunofluorescence in BSR-T7 cells transfected with expression plasmids. Cells were stained with murine anti-L1 antibody and anti-mouse IgG fluorescein isothiocyanate (secondary antibody) (Caltag). (a) pTM-L-AVVA; (b) pTM-L1; (c) pTM1 (empty vector, negative control). 8466 NOTES J. VIROL. Downloaded from jvi.asm.org FIG. 5. Western immunoblot analysis of L-flag epitope tag fusions. Lysates were obtained from BSR-T7 cells transfected with pTM-T⌬C180 (lane 1) (A) or pTM-1 (empty vector) (lane 2); pTM-T⌬N1062 (lane 1) (B), pTM-T⌬C759 (lane 2), pTM-T⌬N394 (lane 3), or pTM-1 (empty vector) (lane 4); or pTM-1 (empty vector) (lane 1) (C) or pTM-TL (lane 2). Blots were probed with anti-flag M2 murine primary antibody (Sigma) ⌬ and goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma). Arrows indicate protein from: pTM-T C180 (a), at COLUMBIA UNIVERSITY on May 21, 2010 pTM-T⌬N394 (b), pTM-T⌬C759 (c), pTM-T⌬N1062 (d), or pTM-TL (e). the SV40 type of core NLS (2). Proline residues are present The results presented here with respect to L and elsewhere flanking the L-NLS; 852P but not 854P seems to play a role in with respect to N, P, and X, confirm the intimate association the nuclear localization of L (pTM-Z⌬N824C853; Fig. 3C, between BDV and the host cell nucleus predicted by Sasaki panel f). and Ludwig (11) and Gosztonyi and Ludwig (7).

FIG. 6. Western immunoblot analysis of ␤-galactosidase-L fusion proteins. Lysates were obtained from BSR-T7 cells transfected with: (A) pTM-1 (empty vector) (lane 1), pTM-ZL (lane 2), pTM-Z⌬C824 (lane 3), pTM-Z⌬C759 (lane 4), pTM-T⌬C180 (lane 5), pTM-Z⌬N1439 (lane 6), pTM-Z⌬N1062 (lane 7), pTM-ZDN824 (lane 8), pTM-Z⌬N561 (lane 9); (B) pTM-1 (empty vector) (lane 1), pTM-Z⌬N824C1062 (lane 2), pTM-Z⌬N921C1062 (lane 3), pTM-Z⌬N921C1015 (lane 4), pTM-Z⌬N854C1015 (lane 5), pTM-Z⌬N824C941 (lane 6), pTM-Z⌬N824C853 (lane 7). Blots were probed with anti-␤-galactosidase murine primary antibody (Sigma) and goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma). VOL. 76, 2002 NOTES 8467

3. Briese, T., J. C. de la Torre, A. Lewis, H. Ludwig, and W. I. Lipkin. 1992. Borna disease virus, a negative-strand RNA virus, transcribes in the nucleus of infected cells. Proc. Natl. Acad. Sci. USA 89:11486–11489. 4. Briese, T., A. Schneemann, A. J. Lewis, Y. S. Park, S. Kim, H. Ludwig, and W. I. Lipkin. 1994. Genomic organization of Borna disease virus. Proc. Natl. Acad. Sci. USA 91:4362–4366. 5. Cubitt, B., and J. C. de la Torre. 1994. Borna disease virus (BDV), a nonsegmented RNA virus, replicates in the nuclei of infected cells where infectious BDV ribonucleoproteins are present. J. Virol. 68:1371–1381. 6. Go¨rlich, D. 1997. Nuclear protein import. Curr. Opin. Cell Biol. 9:412–419. 7. Gosztonyi, G., and H. Ludwig. 1984. Borna disease of horses. An immuno- histological and virological study of naturally infected animals. Acta Neuro- pathol. 64:213–221. 8. Kobayashi, T., Y. Shoya, T. Koda, I. Takashima, P. K. Lai, K. Ikuta, M. Kakinuma, and M. Kishi. 1998. Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein. Virology 243:188–197. 9. LaCasse, E. C., and Y. A. Lefebvre. 1995. Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res. 23:1647–1656. 10. Pyper, J. M., and A. E. Gartner. 1997. Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of

FIG. 7. Western immunoblot analysis of L-NLS mutant. Lysates Borna disease virus. J. Virol. 71:5133–5139. Downloaded from 11. Sasaki, S., and H. Ludwig. 1993. In Borna disease virus infected rabbit were obtained from BSR-T7 cells transfected with pTM-1 (empty neurons 100 nm particle structures accumulate at areas of Joest-Degen vector) (lane 1) or pTM-L-AVVA (lane 2) and probed using murine inclusion bodies. Zentralbl. Veterinarmed. 40:291–297. anti-L1 antisera with goat anti-mouse IgG secondary antibody conju- 12. Schwemmle, M., C. Jehle, T. Shoemaker, and W. I. Lipkin. 1999. Charac- gated to horseradish peroxidase (Sigma). terization of the major nuclear localization signal of the Borna disease virus phosphoprotein. J. Gen. Virol. 80:97–100. 13. Shoya, Y., T. Kobayashi, T. Koda, K. Ikuta, M. Kakinuma, and M. Kishi. 1998.

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