Borna disease virus (BDV) replicates and transcribes its negative-sense RNA genome in the nucleus. The BDV phosphoprotein (P) is localized in the nucleus of infected cells and cells transfected with P expression constructs. To identify the nuclear localization signal (NLS) of P, COS- 7 cells were transfected with wild-type or mutant forms of P fused with green fluorescent protein (GFP). Whereas GFP alone was exclusively cytoplasmic, P or P-GFP were nuclear. Analysis of carboxy- and amino- terminal truncation mutants of P indicated that amino acids (aa) 20-37 are sufficient to promote efficient nuclear accumulation of the fusion protein. Residual nuclear import of GFP was observed with portions of P including aa 33-134 or aa 134-201, suggesting the presence of additional NLS motifs. The major NLS of P appears to be bipartite. It consists of two basic aa domains, R22RER25 and R30PRKIPR36, separated by four non-basic aa, S26GSP29

Jehle, Christian

Lipkin, W. Ian

Schwemmle, Martin

Shoemaker, Trevor

Journal of General Virology

Columbia University Academic Commons

Journal of General Virology (1999), 80, 97–100. Printed in Great Britain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHORT COMMUNICATIONCharacterization of the major nuclear localization signal of theBorna disease virus phosphoproteinMartin Schwemmle,1, 2 Christian Jehle,1 Trevor Shoemaker2 and W. Ian Lipkin21 Abteilung Virologie, Institut fu$ r Medizinische Mikrobiologie und Hygiene, Universita$ t Freiburg, PO Box 820, D-79008 Freiburg, Germany2 Laboratory for Neurovirology and Microbial Pathogenesis, Departments of Neurology, Anatomy and Neurobiology, and Microbiology andMolecular Genetics, University of California–Irvine, Irvine, CA 92697-4292, USABorna disease virus (BDV) replicates and trans-cribes its negative-sense RNA genome in the nu-cleus. The BDV phosphoprotein (P) is localized inthe nucleus of infected cells and cells transfectedwith P expression constructs. To identify the nuclearlocalization signal (NLS) of P, COS-7 cells weretransfected with wild-type or mutant forms of Pfusedwithgreenfluorescentprotein (GFP).WhereasGFP alone was exclusively cytoplasmic, P or P–GFPwere nuclear. Analysis of carboxy- and amino-terminal truncation mutants of P indicated thatamino acids (aa) 20–37 are sufficient to promoteefficient nuclear accumulation of the fusion protein.Residual nuclear import of GFP was observed withportions of P including aa 33–134 or aa 134–201,suggesting the presence of additional NLS motifs.The major NLS of P appears to be bipartite. It con-sists of two basic aa domains, R22RER25 andR30PRKIPR36, separated by four non-basic aa,S26GSP29.Borna Disease virus (BDV) is a non-segmented negative-strand RNA virus that causes persistent central nervous systeminfection and behavioural disturbances in warm-bloodedanimals (Ludwig et al., 1988 ; Rott & Becht, 1995). It encodes atleast six proteins : the nucleoprotein (N), phosphoprotein (P)(Thiedemann et al., 1992 ; Thierer et al., 1992), atypicalglycoprotein (gp18) (Kliche et al., 1994 ; Stoyloff et al., 1994),type I membrane glycoprotein (p57) (Gonzalez-Dunia et al.,1997 ; Schneider et al., 1997), polymerase (pol) and X-protein(Briese et al., 1994 ; Cubitt & de la Torre, 1994 ; Wehner et al.,1997). BDV replicates in the nucleus (Briese et al., 1992 ; Cubittet al., 1994) and employs the cellular splicing machinery for thematuration of some of its viral transcripts (Schneemann et al.,Authors for correspondence: Martin Schwemmle.Fax ›49 761 203 6638. e-mail schwemm!ukl.uni-freiburg.deIan Lipkin (at Laboratory for Neurovirology and Microbial Pathogenesis,3rd Floor Gillespie Building). Fax ›1 949 824 1229.e-mail ilipkin!uci.edu1995). Thus, transport of viral RNAs and proteins betweennucleus and cytoplasm is an essential feature of the BDV life-cycle. A functional nuclear localization signal (NLS) wasrecently described for the N protein (Kobayashi et al., 1998 ;Pyper & Gartner, 1997). Three lines of evidence indicate thatP may also contain an NLS and mediate nuclear import of otherBDV proteins : (1) transient expression of P results in its nuclearaccumulation ; (2) X interacts with P but not N; and (3) thepresence of P in transfected and infected cells shifts thedistribution of X from the cytoplasm to the nucleus(Schwemmle et al., 1998).To identify regions of P that mediate nuclear localizationwe employed a strategy similar to that used to characterize theNLS of lymphoid enhancer factor-1 (Prieve et al., 1996). A setFig. 1. Localization of P, GFP and P–GFP fusion proteins after transientexpression in COS-7 cells. Plasmids encoding BDV-P, P–GFP and GFP, orP–GFP mutants lacking the amino-terminal 32 aa (ND32), 93 aa (ND93)and 133 aa (ND133) or the carboxy-terminal 67 aa (CD67), 107 aa(CD107) or 166 aa (CD166), were transiently expressed in COS-7 cells.The fusion proteins were detected by immunofluorescence after incubationwith antisera to P or GFP.0001-5790 # 1999 SGM JHM. Schwemmle and othersFig. 2. Mapping of the major NLS of P.Plasmids encoding a selection of P–GFPmutants were transiently expressed inCOS-7 cells. Fusion proteins weredetected by immunofluorescence usingantisera to P. Upper panels : carboxy-terminal deletion of 134 aa (CD134),160 aa (CD160) and 164 aa ofP(CD164) of P. Middle panels : carboxy-terminal deletion of 160 aa with anadditional substitution at aa position 32(R32G–CD160), deletion of carboxy-terminal 134 aa and 19 (ND19–CD134)or 27 amino-terminal aa (N27–C134).Lower panels : carboxy-terminal deletionof 160 aa with three aa substitutions(T20S-S26,28A–CD160), amino- andcarboxy-terminal deletion of 32 aa and67 aa (ND32–CD67). See Fig. 3(A) fordetails of plasmid construction.of constructs was generated that encode various portions of Pfused to a modified version of the green fluorescent protein(GFP), a marker protein that is confined to the cytoplasm in theabsence of an exogenous NLS (Prieve et al., 1996). A summaryof constructs and results of experiments described below canbe found in Fig. 3 (A). The corresponding cDNA regions of P[amino acids (aa) 2–201] were amplified from the plasmid P-PTRE (Schwemmle et al., 1998) by PCR and cloned into theBamHI site of the GFP expression vector (Prieve et al., 1996).P wild-type, GFP or P–GFP were expressed in COS-7 cells bytransient transfection using lipofectamine (BoehringerMannheim). After 48 h, the cells were fixed with 4%paraformaldehyde for 10 min and permeabilized with 0–5%Triton X-100. Thereafter, the fusion proteins were detected byimmunofluorescence using either a polyclonal anti-P antibody(Kliche et al., 1996) or a polyclonal anti-GFP antibody(Clontech). Whereas both P and P–GFP accumulated in thenucleus, GFP was present only in the cytoplasm (Fig. 1).Carboxyl- and amino-terminal deletions and point muta-tions were introduced into the P–GFP construct to define theregion(s) of P responsible for the nuclear import of P–GFP.Transient transfection of P–GFP constructs lacking the amino-terminal 32 aa (ND32), 93 aa (ND93) or 133 aa (ND133)resulted in expression of fusion protein in both cytoplasm andnucleus (Fig. 1). In contrast, fusion proteins truncated at thecarboxyl terminus of 67 aa (CD67) or 107 aa (CD107) of P weredetected only in the nucleus (Fig. 1). Deletion of 166 carboxy-terminal aa of P (CD166) resulted in predominantly cytoplasmicstaining (Fig. 1). These experiments indicated that the amino-terminal 35 aa of P are essential but not sufficient to directnuclear accumulation of GFP. Thus, the P constructs CD134,CD160 and CD164 (Fig. 3A) were created to examine theimportance of sequence carboxyl to the first 35 aa of theprotein. Expression of each of these constructs resulted in thenuclear accumulation of the reporter protein (Fig. 2), suggestingthat the nuclear localization function extends to aa 36 (R) and37 (N).The stretch between aa 22 and aa 36 of P includes twobasic regions reminiscent of bipartite NLSs in other systems(Dingwall, 1991) (Fig. 3B). To examine the role of theseregions in nuclear localization we created GFP expressionconstructs encoding aa 20–67 (ND19–CD134) and aa 28–67(ND27–CD134). Whereas ND19–CD134, a fusion proteincontaining the first basic region (R22RER25), was foundpredominantly in the nucleus, ND27–CD134 was present inboth cytoplasm and nucleus, suggesting that aa 20–27 con-tribute to the efficient nuclear accumulation of GFP (Fig. 2). Thesignificance of the central arginine (R32) in the second basicregion was examined by mutating this residue to glycine in aconstruct containing aa 2–41 of P (R32G–CD160). Theresulting mutant fusion protein accumulated less efficiently inthe nucleus indicating that R32 is an important component ofthe NLS. Next, the role of residues within the non-basic regionthat separates the two basic regions of the bipartite NLS wasassessed. Serines 26 and 28, previously identified as sites forphosphorylation by PKCe (Schwemmle et al., 1997), wereJINLS of BDV-PFig. 3. (A) Schematic representation of the P–GFP fusion proteinstransiently expressed in COS-7 cells. The distribution of fusion protein48 h after transfection is indicated as follows: C, cytoplasmic ; N, nuclear ;C/N, cytoplasmic and nuclear. (B) Sequence of the bipartite nuclearlocalization signal of BDV-P. Residues contained in the two basic regionsthat constitute the bipartite signal are indicated by black bars.mutated to alanines, and threonine 20 was mutated to serine(T20S-S26,28A–CD160). These mutations outside of the twobasic domains that constitute the putative bipartite NLS had noimpact on GFP distribution, providing further support for thepresence of a strong bipartite NLS (Fig. 2).The observation that P–GFP fusion proteins truncated atthe amino terminus of 32, 93 or 133 aa (ND32, ND93 andND133) were primarily but not exclusively cytoplasmic (Fig. 1)suggests the presence of weak NLS sequences in portions of Pother than those that constitute the bipartite NLS. To addressthis possibility, a construct was created in which GFP wasfused to aa 33–134 of P (ND32–CD67). Proteins expressedfrom ND32–CD67 (Fig. 2) were present predominantly in thecytoplasm; however, signal was also detected in the nucleus. Inconcert with results obtained with ND133, these data areconsistent with the presence of at least two potential NLSs :one between aa 33 and 134, and a second carboxyl to residue134.The rate of nuclear import of some viral proteins, forexample SV40 large T-antigen (Rihs et al., 1991) and influenzavirus nucleoprotein (Neumann et al., 1997), is dependent onphosphorylation by cellular protein kinases. In studies reportedhere mutation of PKC phosphorylation sites within thebipartite NLS (serines at aa 26 and 28) (Schwemmle et al., 1997)had no impact on the nuclear localization of P–GFP fusionprotein (Fig. 2) ; however, we did not examine either thekinetics of nuclear import in PKC mutants or the possibilitythat phosphorylation might influence the distribution of P inthe presence of other BDV proteins.In contrast to the nucleoprotein of BDV, which is reportedto have a single continuous NLS at the amino terminus(Kobayashi et al., 1998 ; Pyper & Gartner 1997), the major NLSof P appears to be bipartite. Another difference may be thepresence in P of additional weaker NLSs toward the carboxylterminus. The bipartite NLS of P, spanning aa 22–36, overlapsthe region of P essential to interactions with X (aa 33–115)(Schwemmle et al., 1998). The observation that coexpression ofP and X results in colocalization of the two proteins in thenucleus (Schwemmle et al., 1998) is consistent with activity ofweaker NLSs, when P is bound to X ; however, it is equallyplausible that P–X complexes are imported to the nucleusthrough activity of the bipartite NLS. There is precedent ininfluenza virus nucleoprotein (Neumann et al., 1997 ; Wang etal., 1997 ; Weber et al., 1998) and herpes simplex virus type 1ICP27 (Mears et al., 1995) for multiple NLSs. However, wecannot discern from current information whether these NLSsbetween aa 33 and 201 of P are biologically significant or onlypotential NLS motifs that are unmasked through mutagenesisof P. Reverse genetic approaches and analysis of interactions ofP with other viral and host proteins in infected cells will beessential to establish the basis for P trafficking in vivo.This work was supported by Deutsche Forschungsgemeinschaft grantSchw 632}2-1 and National Institutes of Health Grant NS-29425. Wethank Thomas Briese, Peter Sta$ heli and Nigel Horscroft for helpfulcomments.ReferencesBriese, T., de la Torre, J. C., Lewis, A., Ludwig, H. & Lipkin, W. I.(1992). Borna disease virus, a negative-strand RNA virus, transcribes inthe nucleus of infected cells. Proceedings of the National Academy of Sciences,USA 89, 11486–11489.Cubitt, B. & de la Torre, J. C. (1994). Borna disease virus (BDV), anonsegmented RNA virus, replicates in the nuclei of infected cells whereinfectious BDV ribonucleoproteins are present. Journal of Virology 68,1371–1381.Dingwall, C. L. R. (1991). 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Characterization of the major nuclear localization signal of the Borna disease virus phosphoprotein

M Schwemmle

C Jehle

T Shoemaker

W I Lipkin

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Characterization of the major nuclear localization signal of the Borna disease virus phosphoprotein.

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Characterization of the major nuclear localization signal of the Borna disease virus phosphoprotein

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