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 Downloaded by guest on October 2, 2021 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.