Proc. Natl. Acad. Sci. USA Vol. 89, pp. 11486-11489, December 1992 Neurobiology Borna disease virus, a negative-strand RNA virus, transcribes in the nucleus of infected cells (central nervous system infection/behavioral disorders) THOMAS BRIESE*t, JUAN CARLOS DE LA TORREt, ANN LEWIS§, HANNS LUDWIG*, AND W. IAN LIPKIN§¶ *Institute of Virology, Free University of Berlin, Nordufer 20, D 1000 Berlin 65, Federal Republic of Germany; tDepartment of Neuropharmacology, Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037; and IDepartments of Anatomy and Neurobiology, Neurology, and Microbiology and Molecular Genetics, University of California, Irvine, CA 92717 Communicated by D. Carleton Gajdusek, September 1, 1992 (receivedfor review April 22, 1992)

ABSTRACT Borna disease virus, an uncas ied Infec- 100,000 x g for 1 hr at 200C, resuspended in 20 mM Tris HCl, tious agent, causes immune-mediated neurologic disease in a pH 7.4/125 mM MgCl2 (Tris-Mg,2 buffer), and treated with wide variety of animal hosts and may be involved in patho- DNase (Boehringer Mannheim) at 50 Ag/ml and RNase genesis of selected neuropsychiatric diseases in man. 1nitial (Boehringer Mannheim) at 50 ,ug/ml for 1 hr at 3TC. Virus reports suggested that Borna disease virus is a singlesranded particles were pelleted at 100,000 x g for 1 hr at 40C and RNA virus. We describe here a method for isolatin of viral resuspended in Tris-Mg,25 buffer for virus titration or nucleic particles that has allowed definitive identification ofthe genome acid extraction. as containg a negative-polarity RNA. Further, we show that Extraction of RNAs from Virus Partickles. Virus particle the viral mRNAs are transcribed in the nucleus. preparations were mixed with an equal volume of 2x lysis buffer (100 mM EDTA/10%o SDS/2% 2-mercaptoethanol/ Borna disease is an immune-mediated behavioral syndrome 400 pg ofproteinase K per ml/100 mM Tris-HCl, pH 8.3) and caused by infection with an unclassified agent, Borna disease incubated for 45 min at 650C. After cooling to room temper- virus (BDV). Though natural infection has only been con- ature, preparations were extracted twice with phenol/ firmed to occur in horses and sheep, the potential host range chloroform/isoamyl alcohol. The final aqueous phase was of BDV includes other mammals and birds. Experimental supplemented with glycogen to 40 Ag/ml and placed at -200C infection of birds, rodents, and primates leads to the expres- overnight with 1/10th volume of3 M sodium acetate (pH 5.0) sion of BDV proteins in neural tissues, meningoencephalitis, and 1 volume ofisopropyl alcohol. Precipitated nucleic acids and behavioral disturbances (1). Antibodies reactive with were pelleted at 20,000 x g for 30 min at 40( and resuspended BDV proteins have been found in patients with neuropsy- in H20/0.5% SDS for Northern hybridization analysis. Total chiatric diseases, suggesting the possibility that BDV or a cell RNA from rat brain was extracted by homogenization in related virus may be a human pathogen (2-7). guanidinium isothiocyanate and centrifugation through ce- Though the agent has not been identified by electron sium chloride (17). Total cell RNA from tissue culture cells microscopy, molecular approaches have facilitated its partial was extracted with guanidinium isothiocyanate/acid phenol characterization. BDV cDNA clones have been isolated by (18). subtractive screening of libraries prepared from infected rat RNA Nuceocytoplsmic Transport. RNA nucleocytoplas- brain (8) and infected cells (6). Analyses of BDV transcripts mic transport experiments were done according to methods in Northern and in situ hybridization experiments have of Schroder et al. (19). Uninfected or persistently BDV- indicated that BDV is likely to be a single-stranded RNA infected C6 cells (American Type Culture Collection) were virus with a genome of 8.5 (8, 9) to 10 kilobases (kb) (6). The cooled on ice, washed three times with HB buffer (10 mM polarity ofthe viral genome has been controversial (6, 8-11). Tris-HCl, pH 7.5/1 mM MgCl2/1 mM EGTA), and then Here we report that infectious BDV particles contain a homogenized on ice in HB buffer/i mM phenylmethylsulfo- negative-polarity 8.5-kb RNA and demonstrate that viral nyl fluoride using a Dounce homogenizer (40 strokes with an mRNAs are synthesized in cell nuclei. "A" pestle). The suspension was brought to 50%6 (wt/vol) sucrose/20 mM Tris HC1, pH 7.5/1 mM MgCl2 and spun over MATERIALS AND METHODS a 60o (wt/vol) sucrose/20 mM Tris-HCl, pH 7.5/1 mM MgCl2 cushion at 130,000 x g for 1 hr at 40C. Nuclei were Virus Particle Preparation. Oligo/TL, an human oligoden- resuspended in transport buffer (25 mM Tris-HC1, pH 7.5/250 drocyte cell line (12), was infected at an estimated multiplic- mM sucrose/0.5 mM CaCl2/0.3 mM MnCl2/25 mM KCI/5 ity ofinfection of0.5 focus-forming unit per cell using BD rat mM spermidine/5 mM 2-mercaptoethanol/yeast tRNA at 300 brain homogenate (strain V, sixth rat passage) (13-15). Cells jug/ml). Nuclei were resuspended in aliquots (840 Al). Indi- were passed every 2-3 days and used for virus particle vidual aliquots were mixed with 160 1d of either an ATP- preparation in passages 10 through 25. Infectious virus was regenerating system (15.6 mM ATP/31.2 mM Na2HPO4/31.2 titered in a cell-ELISA system (16). The confluent cell layer mM phosphoenolpyruvate/pyruvate kinase at 218 units/ml) (1-2 x 108 cells) was washed once with 20 mM Tris-HCl (pH or the same buffer system without ATP (-ATP control). 7.4), overlaid with 20 mM Tris HCl, pH 7.4/250 mM MgCl2 Transport kinetics were performed at 300C. Reactions were (Tris-Mg250 buffer), and incubated for 1.5 hr at 370C to release stopped on ice at 0, 15, or 40 min and spun at 1000 x g for the cell-bound virus. Supernatant was collected and spun 4 min at 00C. Supernatants (postnuclear fractions) were twice at 2500 x g for 5 min. Clarified supernatant was brought extracted with phenol/chloroform/isoamyl alcohol, placed to 0.002% Zwittergent 3-14 (Calbiochem) and incubated for 1 at -20oC overnight with 1/10th volume of3 M sodium acetate hr at room temperature. Virus particles were pelleted at Abbreviations: BDV, Bornadisease virus; ORF, open readingframe. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Neurology, University of Califor- payment. This article must therefore be hereby marked "advertisement" nia, Irvine, CA 92717. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed. 11486 Neurobiology: Briese et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11487 (pH 5.0) and 2 volumes ofethanol. Precipitated nucleic acids Table 1. Virus particle preparation were pelleted and poly(A)+ RNA was selected by oligo(dT) Virus yield, ffu x 107 chromatography (20). Total RNA from nuclear fractions was extracted with guanidinium isothiocyanate/acid phenol (18). Step Treatment Total* max, mmn Total RNA from nuclear fractions or poly(A)+ RNA from 1 Overlay washed cell layer 57.0 84.0, 12.0 postnuclear fractions was analyzed by Northern hybridiza- (2 x 108 cells) with Tris-Mg2so tion with probes for detection of BDV RNAs or cyclophilin buffer; incubate 1.5 hr, 3rC; mRNA. collect supernatant and spin 5 Probes. 32P-labeled probes for Northern hybridizations min, 2500 x g, RT were prepared by random-hexanucleotide priming ofplasmid 2 Adjust supernatant to 0.002% 2.3 6.6, 0.24t cDNAs (21). 32P-labeled RNA probes were prepared from a Zwittergent; incubate 1 hr, RT plasmid cDNA template according to protocols from 3 Spin 1 hr,100,000 X g, 206C; 2.7 4.0, 1.5 Promega. Plasmids used were pAF4, encoding the 24-kDa resuspend pellet in Tris-Mg,25 protein ofBDV (8), and p1B15, encoding rat cyclophilin (22). buffer Northern Hybridization.-For viral particle analysis, sam- 4 Add DNase, RNase (50pg/ml 2.5 4.2, 1.3 ples were RNA from particles released by 10i Oligo/TL cells each); incubate 1 hr, 37TC or 10 pug of RNA from normal or BDV-infected rat brain and 5 Spin 1 hr, 100,W0 X g, 4C; 4.3 7.3, 1.5 normal or BDV-infected Oligo/TL cells. For RNA nucleo- resuspend pellet in Tris-Mgu5 cytoplasmic transport analysis, samples were total RNA buffer from nuclear fractions or poly(A)+ RNA from postnuclear ffu, Focus-forming units; max, maximum; min, minimum; RT, fractions. RNAs were size-fractionated on 2.2 M formalde- room temperature. hyde/1.0% agarose gels and transferred to nylon membranes *Mean of eight preparations. (MSI). Membranes were hybridized with 32P-labeled DNA tValues after Zwittergent treatment vary, but similar yields were probes or 32P-labeled RNA probes at 650C in 6x SSC (lx obtained in all experiments after removal of detergent by centrifu- SSC is 0.15 M sodium chloride/0.015 M sodium citrate)/5 x gation in step 3. Denhardt's solution (lx Denhardt's solution is 0.02% poly- vinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albu- RNAs (<8.5 kb) in extracts from infected rat brain, no RNAs min)/10 mM EDTA/0.5% SDS/salmon sperm DNA at 100 were detected in the virus particle extract. The sense RNA Ag/ml/yeast tRNA at 100 ,ug/ml for 18 hr and then washed in probe detected only the 8.5-kb RNA in the virus particle and 0.2x SSC/0.2% SDS at 650C for 30 min before autoradiog- rat brain extracts. Identical results were obtained using raphy. Membranes hybridized with RNA probes were ex- oligonucleotide probes antisense (ATGCCACTGCGTTCT- posed to RNase A at 100 ,ug/ml/10 mM Tris HCl, pH 7.4/1 GCCTGATATC) or sense (GATATCAGGCAGAACG- mM EDTA/0.3 M NaCl at 370C for 30 min to reduce CAGTGGCAT) to the mRNA encoding the 38/40-kDa pro- background hybridization to rRNAs. RNA standards tein of BDV (data not shown). (GIBCO/BRL) were used to determine approximate size of BDV RNAs Are Tsribed in the Nuce ofIfected Cels. RNAs. RNA nucleocytoplasmic transport studies were performed to determine sites of BDV transcription. Nuclei isolated from RESULTS uninfected and BDV-infected C6 cells were resuspended in nuclear transport buffer. At 0, 15, and 40 min, reactions were Virus Particle Preparation. Reports that elevated salt con- stopped and RNA was extracted from nuclei (nuclear frac- centrations caused enhanced translation of viral RNAs (23) tion) and supernatant (postnuclear fraction) for analysis by led to treatment of BDV-infected cells with hypertonic me- Northern blot hybridization with a probe representing the dia. This resulted in the unexpected finding that hypertonicity ORF for the 24-kDa protein of BDV. At all time points, induced the release of BDV from infected cells (12). Various nuclear extracts from infected cells contained 8.5-, 3.5-, 2.1-, cell lines and salt conditions were studied. Highest yields of and 0.8-kb BDV RNAs (Fig. 2). The 8.5-kb RNA remained released virus were obtained by treating a BDV-infected confined to the nuclear fraction throughout the course ofthe human oligodendrocyte cell line (Oligo/TL) with 250 mM MgCl2 (data not shown). By this method, 5 x 108 infectious A B particles (focus-forming units) were released from 2 x 108 Oligo/TL cells (Table 1). Mild detergent (0.002% Zwitter- NC [ Vc TV II Ik VA gent), applied to remove excess lipid from the clarified samples, resulted in a transient 1-2 logarithm reduction in virus titer. Independent ofdetergent treatment, a 1 logarithm decrease in titer occurred after the first high-speed centrifu- gation of released virus (data not shown). Treatment of released virus preparations with nucleases did not affect virus titer (Table 1). BDV Particles Contain a Negative-Polarity 8.5-kb RNA. Northern hybridization experiments revealed that the 8.5-kb BDV RNA was protected from nucleases during particle a b preparation (Fig. 1A). A double-stranded probe representing the ORF for the 24-kDa protein of BDV detected several FiG. 1. Nort11hern hybridization analysis of virus particle RNA. BDV-specific RNAs (8.5, 3.5, 2.1, and 0.8 kb) in extracts (A) RNA composition of extracts from a virus particle preparation from infected cells. In contrast, only the 8.5-kb RNA was (Vc), infected Oligo/TL cells (IC), and noninfected Oligo/TL cells detected in the virus particle extract. The polarity of the (NC) analyzed by hybridization using a double-stranded probe rep- resenting the open reading frame (ORE) of the 24-kt4 protein of 8.5-kb RNA derived from virus particle preparations was BDV. (B) Polarity ofRNAs extracted from virus particle preparation investigated in hybridization experiments using single- (Vc) and from infected rat brain (In1) analyzed by hybridization usin stranded RNA probes either antisense or same sense to the single-stranded RNA probes either antisense (b) or same sense (a) to mRNA encoding the 24-kDa protein of BDV (Fig. 1B). the mRNA of the 24-kDa protein of BDV. Positions of 285 and 185 Whereas the antisense RNA probe detected the small BDV rRNA are indicated. 11488 Neurobiology: Briese et A Proc. Nati. Acad. Sci. USA 89 (1992)

-ATP -ATP undetected positive-polarity genomic RNAs in the particles, 0 15 40 40 o 15 40 40 N CN C N C N C N C N C N C N C Northern hybridization experiments described here support A the negative-strand RNA virus model. -OR The advent of a method for isolation of infectious BDV 79:g kb prompted us to attempt direct visualization of particles by -4.4 electron microscopy. We have identified 42-nm, spherical, enveloped particles in virus release preparations (W.I.L. and -2.4 C. Ribak, unpublished work). Although such particles are not -1.4 present in preparations from uninfected cells and are consis- WI tent with the biology of the BDV system (a putative 8.5-kb genome; sensitivity to detergents), these particles have not yet been labeled using antibodies from infected animals. Thus, their significance remains uncertain. B -OR We have suggested that BDV may have a nuclear phase for -9.5 kb transcription and replication (9, 10) because of two observa- -7.5 tions: (i) the 24-kDa and 38/40-kDa proteins of BDV are -4.4 present in the nucleus ofinfected cells (refs. 24 and 25; C. G. Eii -2.4 Hatalski and W.I.L., unpublished work) and (ii) Northern -1.4 and in situ hybridization experiments showed that, whereas .I the smaller BDV RNAs are present in the nucleus and the cytoplasm, the 8.5-kb RNA is confined to the nucleus (9, 10). The nucleocytoplasmic transport studies described demon-

i I strate that BDV transcription occurs in the cell nucleus. C6 C6-BDV Influenza virus is the only animal RNA virus known to replicate and transcribe in the cell nucleus (26, 27). Though FIG. 2. Synthesis ofBDV mRNAs takes place in the cell nucleus. it is a negative-strand RNA virus, it is segmented. There is Nuclei from C6 and C6-BDV cells were isolated and RNA nucleo- presently no evidence that BDV is segmented. Further, BDV cytoplasmic transport assay was performed. At the indicated time sequence analysis has shown similarities only to nonseg- points (0, 15, and 40 min) RNA was isolated from the nuclear(N) and postnuclear (C) fractions and analyzed by Northern blot hybridiza- mented, negative-strand RNA viruses (28). Taken together tion using specific probes for detection of BDV RNAs (pAF4) and these data indicate that BDV may represent a previously cyclophilin mRNA (plBlS). C6 refers to uninfected cells; C6-BDV undefined class of animal RNA virus. refers to infected cells. -ATP indicates results of RNA nucleocy- toplasmic transport assays performed in the absence of ATP (time We thank Kathryn Carbone for the generous gift ofa BDV-infected point 40 min). The same blot was successively hybridized with C6 cell line and Liv Bode, Marylou V. Solbrig, John Holland, and probes pAF4 (B) and piB15 (A). The first probe was stripped by Donald Summers for helpful discussions. Support for this work was boiling the membrane for 20 min in 10 mM Tris HCI, pH 7.5/5 mM provided by National Institutes of Health Grants NS-29425 (T.B., EDTA/0.5% SDS. OR, lane origin. A.L., and W.I.L.) and NS-12428 (J.C.T.), University of California Taskforce on AIDS Grant R91I047 (T.B. and W.I.L.), Bundesminis- kinetics. In contrast, at 15 min the 2.1- and 0.8-kb RNAs were teriumfur Forschung und Technologie Grant BMFT-07017660 (H.L.), present in the supernatant fraction. At 40 min the 3.5-, 2.1-, and Deutsche Forschungsgemeinschaft Grant LU142-1 (H.L.). and 0.8-kb RNAs had accumulated in the supernatant frac- W.I.L. is a recipient of a Young Investigator Award from National tion. BDV RNAs were not detected in extracts from unin- Alliance for Research on Schizophrenia and Depression and a Pew fected C6 cells. As a control for these experiments, filters Scholars Award from the Pew Charitable Trusts. were also hybridized with a probe for cyclophilin mRNA. Transport of BDV mRNAs and cyclophilin mRNA was 1. Ludwig, H., Bode, L. & Gosztonyi, G. (1988) Borna Disease: energy-dependent: in the absence of ATP only a negligible A Persistent Virus Infection of the Central Nervous System (Karger, Basel). amount of mRNA was transported to the postnuclear frac- 2. Amsterdam, J. D., Winokur, A., Dyson, W., Herzog, S., tion. Similar results were obtained in studies performed with Gonzalez, F., Rott, R. & Koprowski, H. (1985) Arch. Gen. persistently infected Madin-Darby canine kidney cells (data Psychiatry 42, 1093-1096. not shown). 3. Rott, R., Herzog, S., Fleischer, B., Winokur, A., Amsterdam, J., Dyson, W. & Koprowski, H. (1985) Science 228, 755-756. 4. Bechter, K., Herzog, S., Fleischer, B., Schuttler, R. & Rott, R. DISCUSSION (1987) Nervenarzt 58, 617-624. Though Borna disease was described >150 years ago, BDV, 5. Bode, L., Riegel, S., Ludwig, H., Amsterdam, J. D., Lange, the agent that causes this disease, remains unclassified. As the W. & Koprowski, H. (1988) Lancet H, 689. 6. VandeWoude, S., Richt, J. A., Zink, M. C., Rott, R., Narayan, virus eluded classical methods for purification, formal proofof O. & Clements, J. E. (1990) Science 250, 1276-1281. an infectious basis for Borna disease and preliminary charac- 7. Bode, L., Riegel, S., Lange, W. & Ludwig, H. (1992) J. Med. terization ofthe agent was accomplished only recently through Virol. 36, 30-315. use of molecular approaches (6, 8, 9, 11, 24). Because strand- 8. Lipkin, W. I., Travis, G. H., Carbone, K. M. & Wilson, M. C. specific hybridization to BDV RNAs obtained from infected (1990) Proc. Natl. Acad. Sci. USA 87, 4184 4188. rat brain or infected tissue culture cells indicated that the 9. de la Torre, J. C., Carbone, K. M. & Lipkin, W. I. (1990) 8.5-kb RNA was opposite in polarity to the smaller RNAs, we Virology 179, 853-856. proposed that BDV was likely to be a negative-strand RNA 10. Carbone, K. M., Moench, T. R. & Lipkin, W. I. (1991) J. virus (8, 9). This hypothesis has been controversial. Others Neuropathol. Exp. Neurol. 50, 205-214. amounts positive- and negative-polarity 11. Richt, J. A., VandeWoude, S., Zink, M. C., Narayan, 0. & reported equivalent of Clements, J. E. (1991) J. Gen. Virol. 72, 2251-2255. RNAs in extracts from infected rat brain and argued against 12. Pauli, G. & Ludwig, H. (1985) Virus Res. 2, 29-33. the negative-strand virus model (6, 11). To determine viral 13. Zwick, W., Seifried, 0. & Witte, J. (1929) Arch. Wiss. Prakt. genome polarity it was essential first to refine a method for Tierheilkd. 59, 511-545. isolation ofviral particles. This has been achieved. Though we 14. Hirano, N., Kao, M. & Ludwig, H. (1983) J. Gen. Virol. 64, cannot exclude the formal possibility that there are additional, 1521-1530. Neurobiology: Briese et al. Proc. Nadl. Acad. Sci. USA 89 (1992) 11489

15. Kao, M., Ludwig, L. & Gosztonyi, G. (1984) J. Gen. Virol. 65, Douglas, J., Milner, R. J. & Sutcliffe, J. G. (1988) DNA 7, 1845-1849. 261-267. 16. Pauli, J., Grunmach, J. & Ludwig, H. (1984) Zentralbl. Veter- 23. England, J. M., Howett, M. K. & Tan, K. B. (1975) J. Virol. inaermed. Reihe B 31, 552-557. 16, 1101-1107. 17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, 24. Thierer, J., Riehle, H., Grebenstein, O., Binz, T., Herzog, S., W. J. (1979) Biochemistry 18, 5294-5299. 18. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, Thiedemann, N., Stitz, L., Rott, R., Lottspeich, F. & Nie- 156-159. mann, H. (1992) J. Gen. Virol. 73, 413-416. 19. Schroder, H. C., Bachmann, M. & Muller, W. E. G. (1989) 25. Bause-Niedrig, I., Pauli, G. & Ludwig, H. (1991) Vet. Immunol. Methods for Investigating Nucleo-Cytoplasmic Transport of Immunopathol. 27, 293-301. RNA (Fischer, New York). 26. Herz, C., Stavnezer, E., Krug, R. M. & Gurney, T. (1981) Cell 20. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 26, 391-400. 1408-1412. 27. Jackson, D. A., Caton, A. J., McCready, S. J. & Cook, P. R. 21. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, (1982) Nature (London) 296, 366-368. 6-13. 28. McClure, M. A., Thibault, K. J., Hatalski, C. G., & Lipkin, 22. Danielson, P. E., Forsspetter, S., Brow, M., Calavetta, L., W. I. (1992) J. Virol. 66, 6572-6577.