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Genomic Organization of Borna Disease Virus

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Genomic Organization of Borna Disease Virus Proc. Natl. Acad. Sci. USA Vol. 91, pp. 4362-4366, May 1994 Neurobiology Genomic organization of Borna disease virus (central nervous system Infection/behavioral disorders/negative-strand RNA viruses) THOMAS BRIESE*t, ANETTE SCHNEEMANN*, ANN J. LEWIS*, YOO-SUN PARK*, SARA KIM*, HANNS LUDWIGt, AND W. IAN LIPKIN**§¶ Departments of *Neurology, *Anatomy and Neurobiology, and Microbiology and Molecular Genetics, University of California, Irvine, CA 92717; and tInstitute of Virology, Freie Universitit Berlin, Nordufer 20, D 13353 Berlin, Germany Communicated by Hilary Koprowski, January 27, 1994 ABSTRACT Borna disease virus is a neurotropic negative- RNA. The 5'-terminal sequence from each library was used strand RNA virus that infects a wide range ofvertebrate hosts, to design an oligonucleotide primer for construction of the causing disturbances in movement and behavior. We have next library. cloned and sequenced the 8910-nucleotide viral genome by DNA Sequencing and Sequence Analysis. Plasmid DNA was using RNA from Borna disease virus particles. The viral sequenced on both strands by the dideoxynucleotide chain- genome has complementary 3' and 5' termini and contains termination method (13) using a modified bacteriophage T7 antisense information for five open reading frames. Homology DNA polymerase (Sequenase version 2.0; United States to Filoviridae, Paramyxoviridae, and Rhabdoviridae is found Biochemical). Five to 10 independent clones from each in both cistronic and extracistronic regions. Northern analysis library were sequenced with overlap so that each region of indicates that the virus transcribes mono- and polycistronic the genomic RNA was covered by at least 2 clones. Four RNAs and uses terminatlon/polyadenylylation signals remi- libraries were analyzed, yielding =8.9 kb of continuous niscent ofthose observed in other negative-strand RNA viruses. sequence. Nucleic acid sequence was analyzed with a se- Borna disease virus is likely to represent a previously unrec- quence-analysis software package (Genetics Computer ognized genus, bornaviruses, or family, Bornaviridae, within Group, Madison, WI). Data base searches for related se- the order Mononegavirales. quences and multiple sequence alignments were performed with FASTA and PILEUP. Sequence Determination at the 3' and 5' Termine of BDV Borna disease virus (BDV) is a neurotropic virus that causes Genomic RNA. Genomic RNA from one virus particle prep- an immune-mediated syndrome resulting in disturbances in aration (1-2 x 108 cells) was treated with tobacco acid movement and behavior. Originally described as a natural pyrophosphatase (Epicentre Technologies, Madison, WI) infection of horses in southern Germany (1), Borna disease and circularized with T4 RNA ligase (New England Biolabs) has now been described in sheep (2), cats (3), and domestic (14). The ligated RNA was reverse transcribed with Super- fowl (4). Though natural infection has not been reported in Script II (GIBCO/BRL) using a primer, 5'-GCCTCCCCT- primates, subhuman primates can be infected experimentally TAGCGACACCCTGTA-3', complementary to a region 465 (5, 6). Antibodies to BDV proteins have been found in nt from the 5' terminus of the BDV genome. A 2-,ul aliquot patients with neuropsychiatric disorders (7-9), suggesting of the reverse transcription reaction was used to amplify the that BDV or a related agent may be pathogenic in humans. ligated region by the PCR using Stoffel fragment (Perkin- Because BDV grows only to low titer, it was difficult to Elmer/Cetus). Primers used in the first round of PCR were purify for analysis. However, the identification of BDV 5'-GCCTCCCCTTAGCGACACCCTGTA-3' and 5'-GAAA- cDNA clones by subtractive hybridization (10, 11) and, more CATATCGCGCCGTGAC-3', located 241 nt from the 3' recently, the advent of a method for isolation of virus terminus of the BDV genome. Amplified products were particles (12) led to partial characterization of BDV as a subjected to a second round of PCR using a nested set of negative-strand RNA virus which transcribes its RNA in the primers: 5'-TACGTTGGAGTTGTTAGGAAGC-3', 251 nt cell nucleus (12). We have now cloned and sequenced ge- from the 5' terminus, and 5'-GAGCTTAGGGAGGC- nomic RNA from virus particles. The genomic organization TCGCTG-3', 120 nt from the 3' terminus. PCR products were of BDV indicates that it is likely to represent a distinct virus cloned (15) and sequence across the 5'/3' junction was genus or family within the order Mononegavirales. determined from five independent isolates. Northern Hybridization. Poly(A)+ RNA extracted from MATERIALS AND METHODS acutely infected rat brain by using the FastTrack system (Invitrogen) was size-fractionated in 0.22 M formalde- BDV cDNA Library Preparation and Screening. Genomic hyde/1% agarose gels (16), transferred to Zeta-Probe GT RNA template for library construction was obtained from an nylon membranes (Bio-Rad), and hybridized with random- oligodendrocyte cell line (Oligo/TL) acutely infected with primed 32P-labeled restriction fragments (17) representing BDV strain V (12). For the first genomic library, RNA from open reading frames (ORFs) across the BDV genome (see one virus particle preparation was polyadenylylated with Fig. 4b). RNA transfer, hybridization, and washing were poly(A) polymerase (GIBCO/BRL) to facilitate cloning from performed with the manufacturer's protocol (Bio-Rad). the 3' terminus by oligo(dT)-primed cDNA synthesis. Librar- ies were prepared in pSPORT with the SuperScript plasmid system (GIBCO/BRL). The first library was screened with RESULTS pAB5 and pAF4 radiolabeled restriction fragments (10). Sequencing of Genomic BDV RNA. Beginning from the 3' Subsequent libraries were screened with radiolabeled restric- terminus, a series of four overlapping cDNA libraries was tion fragments from locations progressively 5' on the genomic Abbreviations: BDV, Bornadisease virus; ORF, open reading frame. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" IlThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U04608). 4362 Downloaded by guest on September 25, 2021 Neurobiology: Briese et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4363 constructed by using BDV particle RNA (12) as template. three small ORFs, each with a coding capacity of <16 kDa Previous studies have shown that the genomic RNA is not (Fig. lb). polyadenylylated (18). Thus, to construct the first library, Homology Analysis of Coding Sequence. Predicted amino genomic RNA was polyadenylylated in vitro to facilitate acid sequence for the identified ORFs was used to examine oligo(dT)-primed cDNA synthesis. For the subsequent three data bases for similarity to other proteins. Previous analysis libraries, genome-complementary oligonucleotide primers of the ORF encoding p40 had revealed distant sequence were designed based on 5'-terminal sequence determined in similarity to L proteins of Paramyxoviridae and Rhabdovir- the previous round ofcloning. Each region ofthe genome was idae (19). FASTA analysis of translated sequence from ORFs sequenced by using a minimum of two independent clones. encoding p23, gpl8, and p57 showed no apparent similarity to To determine the sequences at the termini, genomic RNA other viral sequences; however, the p180 ORF sequence was circularized and sequenced across the junction by using consistently retrieved L polymerases of Paramyxo- and Rhabdoviridae. Alignment of the p180 ORF sequence with five independent clones. the sequences of RNA-dependent RNA polymerases of neg- The 8910-nt BDV genome contained antisense information ative-strand RNA viruses showed conservation of both se- for five major ORFs flanked by 53 nt of noncoding sequence quence and linear order of regions homologous among these at the 3' terminus and 91 nt of noncoding sequence at the 5' proteins. Extensive conservation was found in the four terminus (Fig. 1). In3'-5' order, the first two ORFs encoded characteristic motifs for L polymerases of negative-strand two previously described viral proteins, p40 (19) and p23(20). RNA viruses (A-D in Fig. 2) (22, 23). With the exception of The third, fourth, and fifth ORFs had coding capacities of 16 the glycine residue in motif B (position 322 of the alignment), kDa (gpl8), 57 kDa (p57), and 190 kDa (p180), respectively conservation was found for the individual amino acid resi- (Fig. la). Predicted amino acid sequence for the 16-kDa ORF dues postulated to participate in polymerase function (22). correlated with microsequence data for an 18-kDa BDV Conservation was also found for a motif (a in Fig. 2) proposed glycoprotein (S. Kliche, T.B., and W.I.L., unpublished to participate in template recognition (23, 24). The alignment data), originally described as the Borna disease-associated produced by the Genetics Computer Group's PILEUP pro- 14.5-kDa protein (21). The first three ORFs showed no gram placed the p180 ORF sequence between polymerases of overlap and were in frame with the fifth ORF (Fig. lb). The Paramyxo- and Rhabdoviridae. This intermediate position is 57-kDa ORF was in a +1/-2 frame relative to the other four reflected by the presence of conserved amino acids which are ORFs and overlapped the adjacent ORF for gpl8 by 28 in agreement with either the rhabdo- or the paramyxovirus codons and ORF p180 by 34 codons. All ORFs were located sequences (* or x, respectively; Fig. 2). The distance on the positive strand, complementary to the genomic RNA. between conserved motifs a and A was found to be short in ORF analysis of the genomic (negative) strand showed only BDV as it is in rhabdoviruses, whereas this region is highly variable in length and sequence among paramyxoviruses (23). a p40 p23 gpl8 (p57) (p180) The PILEUP-generated dendrogram, obtained by using com- [1110) [603] [426] [1509] [5175] plete p180 ORF and L-protein sequences, indicated that the 3' 5' putative BDV polymerase was more closely related to L polymerases of Rhabdoviridae than to those of Paramyxo- 53 79 10 55 viridae (data not shown).
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