Mented, Negative-Strand RNA Virus

ウイルス 45(2),165-174,1995

特集ネガティブ鎖RNAウイルス

4. The atypical strategies used for gene expression of Borna disease virus, a nonsegmented, negative-strand RNA virus.

ANETTE SCHNEEMANN,* PATRICK A. SCHNEIDER,*•˜ and W. IAN LIPKIN*•˜ *Laboratory for Neurovirology , Department of Neurology, Department of Anatomy and Neurobiology, § Department of Microbiology and Molecular Genetics , University of California-Irvine, Irvine, CA 92717

$ To whom correspondence should be addressed . FAX : (714) 824-2132. Email : ilipkin @ uci. edu

al., 1992), has opened the fields of BDV biology and Abstract pathogenesis for rigorous investigation. BDV has Borna disease virus (BDV) is a neurotropic agent now been defined as an enveloped, nonsegmented that causes disturbances in movement and behavior negative-strand RNA virus (Briese et al., 1992 ; in vertebrate host species ranging from birds to Compans et al., 1994 Zimmermann et al., 1994) and primates. Although the virus has not been isolated its sequence has been determined independently in from human subjects, there is indirect evidence to two laboratories (Briese et al., 1994 ; Cubitt et al., suggest that humans with neuropsychiatric dis- 1994b). Although aspects of gene organization and orders may be infected with BDV. Recently, virus deduced protein sequence suggest relatedness to particles have been isolated and the viral genomic members of the order Mononegavirales (paramyx- RNA has been cloned. This analysis revealed that oviruses, rhabdoviruses and filoviruses), BDV BDV is a nonsegmented, negative-strand RNA shows unusual features such as RNA splicing virus. Unusual features such as RNA splicing, over- (Schneider et al., 1994 ; Cubitt et al. 1994c) and lap of transcription units and transcription signals, overlap of transcription units and transcription as well as sequence dissimilarity for four of five signals (Schneemann et al., 1994). These features major open reading frames to genes of other nonseg- indicate that BDV is likely to represent a previously mented, negative-strand RNA viruses suggest that unrecognized genus or family within this order. BDV is likely to represent a new taxon within the Originally considered to be a natural infection of order Mononegavirales. horses and sheep in Southeastern Germany, infection has now been reported in cats, cattle and birds Introduction in Europe, North America and Africa. (Lundgren The neurologic syndrome known as Borna dis- and Ludwig, 1993 ; Kao et al., 1993 ; Malkinson et ease (BD) was first described approximately 200 al., 1993 ; Caplazi et al., 1994). It is not known years ago (Abildgaard, 1785). The agent respon- whether this apparent extension in host and geo- sible for the disease, Borna disease virus (BDV), graphic range represents improved survey methods remained elusive for many years due to its low level or spread of BDV to new species and locations. The of replication in infected hosts. The identification of potential host range is still larger. In addition to the BDV cDNA clones by subtractive hybridization natural hosts already described, rodents, rabbits and (Lipkin et al., 1990) and more recently the advent of primates are readily infected experimentally a method for isolation of virus particles (Briese et (Ludwig et al., 1988). Whether BDV naturally 166 ANETTE SCHNEEMANN,* PATRICK A. SCHNEIDER,*•˜ and W. IAN LIPKIN*•÷•˜•ö

infects humans is controversial. Serum antibodies to 1989 ; Carbone et al., 1991). BDV have been detected in patients with depression Genomic organization or schizophrenia and monocytes from some of these patients were found to contain viral proteins (Bode Two laboratories have recently cloned and se- et al., 1988 ; Fu et al., 1993 ; Rott et al., 1985). quenced BDV genomic RNA using template from However, there are no confirmed reports of virus ribonucleoproteins (strain He-80) (Cubitt et al., isolation from humans and thus the question of 1994a, 1994b) or virus particles (strain V) (Briese whether BDV can be implicated in human disease et al., 1994). Each reported a genome of approxi- remains open. mately 8,900 nt with complementary termini, encod- Infection results in an immune-mediated progres- ing 5 major open reading frames (ORF's). ORFs I, sive movement disorder (Narayan et al., 1983 ; II, III, IV and V identified by Cubitt et al. are Solbrig et al., 1994, 1995). This disorder has been similar to ORFs p40, p23, gpl8, p57 and p180 (pol) linked in a rat model of experimental infection to identified by Briese et al., respectively, with the disturbances in brain dopamine (DA) systems following exceptions : (1) ORFs I, II, III, IV and V (Solbrig et al., 1994, 1995). Early in disease, animals are in frame with one another whereas p57 is in a + show hyperactivity and exaggerated startle 1/-2 frame relative to the other ORFs in Briese et responses. Later, they begin to display repetitive al.'s sequence ; (2) ORF IV predicts a protein of 40 stereotyped motor behaviors, unusual postures of kDa vs. the 57 kDa protein predicted by ORF p57. limbs and trunk as well as self-mutilation. These These differences have now been reconciled to features are reminiscent of human neuropsychiatric confirm the genomic organization proposed by conditions associated with DA abnormalities like Briese et al. for both viral strains (Cubitt et al., 1994- schizophrenia, post-encephalitic Parkinsonism and c). tardive dyskinesia. Infected animals show enhanced Gene products have only been identified for ORFs behavioral sensitivity to drugs that serve as direct p40 (I), p23 (II) and gpl8 (III) (Mc Clure et al., or indirect agonists of DA receptors (DA 1992 ; Thierer et al., 1992 ; Kliche et al., 1994, agonists) ; conversely the movement and behavior Schadler et al.,1985) . The position of ORFs p40 and disorder is improved following treatment with drugs p23 on the antigenome and features such as mol. wt., that selectively block some classes of DA receptors charge and abundance of the respective proteins in (DA antagonists) (Solbrig et al., 1994, 1995). Still infected cells, as well as the presence of posttrans- later in disease, chronically infected animals may lational modifications (p23 is phosphorylated) become blind from retinopathy, or obese due to (Thiedemann et al.,1992) , suggest that p40 encodes hypothalamic disturbances. As in infections with the viral nucleoprotein (N) and p23 the phospho- other neurotropic agents, such as rabies virus or protein (P). The gpl8 (III) ORF encodes a 18 kDa herpes simplex virus, inoculation into a limb results glycoprotein (Kliche et al.,1994) , while the equiva- in centripetal transsynaptic transport to the CNS lent ORF in other nonsegmented, negative-strand (Carbone et al.,1987) . Olfactory inoculation is more RNA viruses directs synthesis of a non-glycosylated efficient and is presumed to be the route of natural matrix protein (M) (Banerjee, 1991). Glycosylated infection. Expression of the movement and behavior M proteins that resemble gpl8 in size and pI (pI 10) disorder is dependent upon an intact host immune have also been found in coronaviruses, which syn- response since immunosuppression prevents these thesize a 20 kDa glycoprotein with a pI of 10 disturbances in adult-infected animals (Narayan et (Armstrong et al., 1984). There is preliminary evi- al., 1983). Animals infected as neonates do not dence that gpl8 is present in the virus envelope and mount a cellular immune response to the virus but that it may serve as a viral attachment protein develop a disease characterized by hyperactivity (Kliche et al.,1994) . Computer analysis of ORF p57 and subtle learning disturbances (Dittrich et al., (IV) revealed 14 potential N-glycosylation sites as The atypical strategies used for gene expression of Borna disease virus, a nonsegmented, negative-strand RNA virus. 167 well as N-terminal and C-terminal hydrophobic least six primary, polyadenylated RNAs with appar- anchor domains (Briese et al.,1994) . Therefore, it is ent lengths of 0.8 kb, 1.2 kb, 1.9 kb, 2.8 kb, 3.5 kb likely that this gene directs expression of the BDV and 7.1 kb (Briese et al., 1994, Cubitt et al., 1994b) . glycoprotein (G). The most 3' ORF on the viral An additional RNA of 4.7 kb has been reported by antigenome, pol (V), encompasses more than half Cubitt et al. The abundance of these RNAs in infect- of the genome and contains motifs considered criti- ed cells and tissues is consistent with the 3'-to-5' cal to viral RNA polymerase activity (Briese et al., transcriptional gradient found for other nonseg- 1994 ; Cubitt et al., 1994b). Cubitt et al. predicted a mented, negative-strand RNA viruses. Two of the protein of 170 kDa from ORF V. However, the primary transcripts, the 7.1 kb RNA and the 2.8 kb presence of conserved sequences between strain V RNA, have been shown to be posttranscriptionally and strain He80 upstream of the AUG colon of ORF modified by RNA splicing to yield at least two V, as well as analysis of viral transcripts (see additional RNA species, 6.1 kb and 1.5 kb in length below) suggests that pol is expressed from two (Schneider et al., 1994 ; Cubitt et al., 1994c). separate exons to yield a protein of 190 kDa. Addi- BDV subgenomic RNAs were initially mapped to tional smaller ORFs with coding capacities of less the antigenome by Northern blot hybridization in than 16 kDa have been identified on both the posi- two separate laboratories (Briese et al., 1994 ; tive (antigenomic) and negative (genomic) RNA Cubitt et al., 1994b) (figure 1). This analysis strands (Briese et al., 1994 ; Cubitt et al., 1994b). revealed a complex pattern of overlapping tran- Whether any of these ORFs is expressed is un- scripts that included several polycistronic RNAs. known. The results obtained by the two groups differed with respect to the location of the 2.8 kb RNA, the Transcription of the BDV genome 1.5 kb RNA and 6.1 kb RNA. Cubitt et al. reported The BDV genome is transcribed in the nucleus of the 5' end of the 2.8 kb RNA to be coterminal with infected cells (Briese et al., 1992 Cubitt et al., 1994 the 5' end of the 0.8 kb RNA, whereas Briese et al. a) . This is an unusual feature for a nonsegmented, found that it is coterminal with the 7.1 kb RNA. negative-strand animal virus, but has also been Furthermore, Briese et al. found that the 1.5 kb and observed for some plant rhabdoviruses (Jackson et the 6.1 kb RNAs initiate and terminate at the same al., 1987). Other negative-strand viruses known to positions as the 2.8 kb and 7.1 kb RNAs, respective- use the nucleus for transcription and replication ly, but contain approx. 1.3 kb internal deletions not include the influenza viruses whose genomes are present in the larger RNAs. Cubitt et al. did not composed of eight segments of ssRNA (Kingsbury, describe these internal deletions and suggested that 1991). It is not clear what aspects of transcription the 1.5 kb and the 6.1 kb RNAs initiated 1.1 kb and and/or replication dictate the nuclear localization 1.2 kb downstream from the initiation sites of the for the plant rhabdoviruses, but influenza viruses 2.8 kb and the 7.1 kb RNAs, respectively, but were have been shown to require both host cell mRNAs otherwise collinear with the larger RNAs. to prime transcription initiation ("cap stealing") The precise transcription initiation and termina- and host cell splicing factors for post-tran- tion sites were subsequently determined by purifica- scriptional processing of their primary RNA tran- tion of BDV subgenomic RNAs and examination of scripts (Kingsbury, 1991). BDV does not appear to the terminal sequences by RNA circularization and employ cap stealing to initiate gene transcription RT-PCR over the ligated ends (Schneemann et al., (Schneemann et al., 1994) ; however, it does require 1994). This analysis confirmed the transcriptional the cellular splicing machinery to process some of map established by Briese et al. (1994) and showed its primary RNA transcripts (Schneider et al., that the genome contains three transcription initia- 1994 ; Cubitt et al., 1994c). tion sites and four termination sites. The results BDV transcription results in the synthesis of at obtained by direct terminal sequencing suggested 168 ANETTE SCHNEEMANN,* PATRICK A. SCHNEIDER,*•˜ and W. IAN LIPKIN*•÷•˜•ö

Figure 1. Genomic organization and transcriptional map of BDV. The BDV genome is shown as a solid line in 3' to 5' direction. Coding regions for BDV proteins and their respective frames on the antigenome are shown as boxes at the top. The numbers above the boxes indicate the position of the first AUG colon of each open reading frame using the sequence of BDV strainV as a reference. The positions of RNA transcription initiation sites on the genome of BDV strainV are shown as arrows pointing downstream. Transcription termination sites and splice sites are shown as downward vertical lines. Stippled lines indicate that readthhrough at termination sites T2 and T3 results in longer RNAs terminating at T3 and T4, respectively. The 1.2 kb RNA and the 0.8 kb RNA have been shown to be used as mRNAs for the translation of p40 and p23, respectively. p23 could also be translated from the 3.5 kb RNA. Transcripts that might be used for the translation of gpl8, p57 and pol are indicated. It is not known whether the 1.9 kb RNA and the 1.4 kb RNA are used for the translation of BDV proteins.

different sites than those proposed for transcription 1). A semiconserved, U-rich motif that is partially initiation by Cubitt et al. (1994b). Specifically, the copied into the respective transcripts was identified sites of transcription initiation mapped to nu- at sites S1, S2 and S3 (figure 2). This motif is cleotides (nt) 1, nt 43 (S1), nt 1175 (S2) and nt 1885 present in all BDV strains for which genomic (S3) using nucleotide positions of strain V (figure sequence is available (strain He80, strain He80-1 The atypical strategies used for gene expression of Borna disease virus, a nonsegmented, negative-strand RNA virus. 169

Figure 2. Comparison of nucleotide sequences at transcription initiation- and termination sites on the BDV genome. The sequences, shown in 3' to 5' direction, are aligned for maximal similarity. The arrow indicates the position at which cRNA synthesis begins within the transcriptional start signal. The nucleotide sequences and the numerical assignations are from the complete sequence of BDV strainV (Briese et al., 1994). Data for start and stop sites are taken from Schneemann and co-workers (1994).

and strain V) suggesting that it has a functional showing the intergenic region typically observed in role, presumably as a transcription initiation signal. nonsegmented, negative-strand RNA viruses. Gene The motif appears to be specific for BDV in that overlap has been observed previously in respiratory similar sequences are not present at the gene start syncitial virus, a paramyxovirus. In this virus, the sites of other nonsegmented, negative-strand RNA start site for the polymerase gene is located 68 nt viruses. Direct terminal sequencing confirmed the upstream of the termination signal for the 22K gene four transcription termination sites proposed by (Collins, 1991) and it has been proposed that this Briese et al. (1994) and Cubitt et al. (1994b) : nt arrangement serves as a mechanism to attenuate 1192 (T1), nt 1882 (T2), nt 4511 (T3) and nt 8855 expression of the polymerase protein. However, in (T4) (figure 1). Each termination site consists of 6 BDV, the 1.2 kb and the 0.8 kb RNAs are the most to 7 U residues preceded by a single A residue abundant RNAs in infected cells, implying that the (figure 2). This consensus sequence is reminiscent overlap does not significantly affect transcription of of the transcription termination signals in rhab- the respective genes. It is possible that the degree of doviruses and paramyxoviruses. In these virus fam- overlap is a function of the length by which the two ilies, the stretch of U-residues is believed to cause transcription units overlap. If so, a stretch of 18 nt polymerase stuttering, which results in the synthesis may not be sufficient to cause a noticeable decrease of a poly (A) tail at the 3' end of the viral transcripts in transcription of the 0.8 kb RNA. However, until (Banerjee, 1991). It is likely that the U residues in the mechanistic details of polymerase function are BDV have an analogous function. better understood, this remains speculative. One of the more striking features of the BDV The second and third transcription units (0.8 kb genome organization is the unusual constellation of and 2.8 kb/7.1 kb RNAs, respectively) are separat- transcription termination and initiation signals at ed by only 2 nt. Interestingly, the transcription initi- the gene junctions (Schneemann et al., 1994). The ation signal for the 2.8 kb RNA (S3) extends transcription initiation site for the 0.8 kb RNA is upstream across the intergenic region into the termi- located 18 nt upstream of the termination site of the nation signal of the 0.8 kb RNA (T2), such that T2 1.2 kb RNA. Thus the two genes overlap instead of is completely contained within S3 (figure 2). This 170 ANETTE SCHNEEMANN,* PATRICK A. SCHNEIDER,*•˜ and W. IAN LIPKIN*•÷•˜•ö arrangement contrasts with the organization of the First, in contrast to the 1.2 kb RNA, the 1.9 kb rhabdovirus and paramyxovirus gene junction, RNA initiates at the extreme 3' end of the BDV which can usually be divided into three separate genome, which does not contain the consensus domains : transcription termination signal, inter- sequence observed at 51, 52 and 53 (Schneemann et genic region, and transcription initiation signal al.,1994) . In addition, the 1.9 kb RNA is not capped (Banerjee, 1991). The overlap of these domains in and not fully polyadenylated : it contains only 8 to BDV does not appear to interfere with their recogni- 9 adenylate residues at the 3' end, seven of which are tion by the BDV polymerase since termination and encoded by the termination signal (Schneemann et initiation occur efficiently at this gene junction. It is al., 1994). These observations suggest that the 1.9 not clear how the polymerase recognizes the over- kb RNA represents an analog of a leader-contain- lapping domains as separate entities but it is pos- ing RNA similar to those observed for rhabdovir- sible that there are additional sequences upstream uses and paramyxoviruses. In these virus families, of the termination site that prepare actively tran- transcription of the sequence at the 3' end of the scribing polymerase for termination. It will be of genome usually results in formation of a short considerable interest to elucidate the mechanistic leader RNA that terminates upstream of the first details of RNA synthesis at this gene junction since gene at a sequence similar to a transcription termi- it promises new insights into the mechanisms for nation signal (Banerjee, 1991). Although no such regulation of gene transcription in nonsegmented, short leader RNAs have yet been found in BDV negative-strand RNA viruses. -infected cells , analysis of the genomic sequence The transcriptional map of BDV contains several upstream of the start of the first transcription unit polycistronic RNAs that arise from readthrough at revealed a motif located within start signal 51 that the various termination sites. Transcriptional is very similar to the consensus termination signal readthrough is not uncommon in nonsegmented, 3' AUUGUUUU (nt 34-42). It is possible that this negative-strand RNA viruses, however, it is usually signal is a weak terminator due to the exchange of considered to be aberrant and the biological signifi- a G residue for a U residue at the fourth position. cance of the readthrough products is unknown RNA synthesis initiating at the extreme 3' end of (Banerjee, 1991). In contrast, the ability of the BDV the genome might therefore proceed into the adja- polymerase to continue transcription beyond a ter- cent gene to result in the synthesis of a leader mination signal is critical for viability of the virus. -containing RNA . There is precedent for such The 7.1 kb RNA and its splice products are the only leader-containing RNAs in measles virus, a par- RNAs that contain the pol ORF and they are gener- amyxovirus. Measles virus leader-containing tran- ated by readthrough at termination site T3. Tran- scripts are functionally distinct from their leader- scriptional readthrough may provide a mechanism less counterparts in that they are not translated but for regulating BDV gene expression. For example, preferentially encapsidated into ribonucleoprotein low level readthrough at T3 would lead to a de- complexes (Castaneda and Wong, 1990). creased level of the BDV polymerase protein, which It is intriguing that the 1.9 kb RNA is not should be needed only in catalytic amounts. Support polyadenylated to the same extent as the other for this hypothesis comes from the observation that subgenomic RNAs (Schneemann et al.,1994) . From the levels of the 7.1 kb RNA and its splice products the perspective of the transcription-replication are indeed lower than those of the 2.8 kb RNA and model of nonsegmented, negative-strand RNA vir- its splice products. It is possible, however, that this uses, the 1.9 kb transcript could also be considered observation merely reflects a difference in the sta- a replication intermediate that was aborted at ter- bilities of the respective RNAs. mination site T2. If this model is correct, the The 1.9 kb RNA was found to be fundamentally polymerase would not be expected to synthesize a different from the other subgenomic BDV RNAs. poly (A) tail at the 3' end of the newly synthesized The atypical strategies used for gene expression of Borna disease virus, a nonsegmented, negative-strand RNA virus. 171

RNA strand. gesting that p40 and p23 are not essential to splicing The BDV genome is extremely compact : 99.4% of the 2.8 kb RNA. of its nucleotides are transcribed into subgenomic Northern blot analysis of RNA isolated from RNAs (Schneemann et al., 1994). Only 55 out of BDV-infected rat brain tissues shows that splicing 8910 bases (strain V) are not found in the primary is not 100% efficient. Roughly equal proportions of viral transcripts. These bases represent the trailer spliced and unspliced messages are present in infect- region at the 5' end of the genome. The region ed cells. This low splicing efficiency is likely to be between the 3' end of the genome and the first base critical to BDV biology as both spliced and uns- of the first transcription unit is 42 nt long and pliced transcripts are predicted to function as probably corresponds to the leader sequence found mRNAs in their own right and to encode distinct in other nonsegmented, negative-strand RNA vir- proteins (see below). The low splicing efficiency uses. Two other bases located in the intergenic may be due to decreased recognition of the 3' splice region between T2 and S3 are found only in the rare sites where BDV RNAs deviate slightly from the polycistronic 3.5 kb RNA. mammalian consensus sequence. Fifty-five percent of all mammalian 3' exons start with a G residue Posttranscriptional modification of BDV transcripts (Krawczak et al., 1992) while BDV 3' exons start Several observations suggested that BDV tran- with either a U residue (exon-2) or a C residue scripts might be modified by RNA splicing. These (exon-3). A decrease in the efficiency of splicing, included (1) the nuclear localization of BDV tran- presumably due to inaccessibility of the 3' splice site scription, (2) the apparent absence of mRNAs initiat- to components of the splicing machinery, has also ing and terminating close to the start and stop sites been reported in influenza virus (Plotch and Krug, of the p57 and pol ORFs and (3) the existence of 1986). transcripts that are not collinear with the BDV Regulation of BDV protein expression genome. That splicing does indeed occur was subsequently confirmed through RT-PCR and Northern Only two primary monocistronic mRNAs have hybridization analysis of RNAs from infected tis- been identified among BDV transcripts : the 1.2 kb sues and cultured cells (Schneider et al., 1994 ; RNA, which represents the mRNA for p40 (McClur- Cubitt et al., 1994c). Two BDV transcripts, the 7.1 e et al., 1992), and the 0.8 kb RNA, which represents kb RNA and the 2.8 kb RNA, were each found to the mRNA for p23 (Thierer et al., 1992). The lack contain two introns that span nt positions 1932-2025 of primary monocistronic RNAs for the gpl8, p57 (intron-l, 94 nt) and 2410-3703 (intron-2, 1.3kb) and poi ORFs and the synthesis of transcripts that (figure 1). Sequence analysis revealed that 78% of contain all three ORFs suggests that posttran- the positions in the 5' and 3' splice sites of both scriptional modifications are necessary for the introns matched the mammalian splice site consen- expression of the downstream ORFs, p57 and poi. sus sequence. In addition, similar to mammalian The most reasonable interpretation of the data RNAs, branchpoint nucleotides are present 18 nt obtained to date, considering the position of the (intron-1) and 30 nt (intron-2) from the splice site coding sequences on the genome, the splicing of the junctions (Schneider et al., 1994). To determine mRNAs and the "context" of the various possible whether these viral RNAs could serve as substrates AUG initation colons (Kozak, 1989), is shown in for host spliceosomes, a truncated cDNA clone of figure 1. For gpl8, the translation initiation colon the 2.8 kb RNA was transiently transfected into has been identified ; it is located 8 nt downstream of COS-7 cells. This construct did not encode the two the 5' end of the 2.8 kb/7.1 kb RNAs. This short viral proteins already known to localize to the untranslated region is likely to result in inefficient nucleus, p40 and p23. Nonetheless, the transfected translation initiation by ribosomal preinitiation cells synthesized the predicted splice products sug- complexes and might serve as a mechanism to 172 ANETTE SCHNEEMANN,* PATRICK A. SCHNEIDER,*•˜ and W. IAN LIPKIN* •÷•˜•ö downregulate gpl8 expression. Splicing of intron-1 der bequemsten and wohlfeilesten Art sie zu from either the 2.8 kb or 7.1 kb RNA effectively heilen. Zum Gebrauch des Landmanns. (J. T. Edlen von Trattnern, Ed . ) , Wien. eliminates the gpl8 ORF by removing 23% of its 2) Armstrong, J., Niemann, H., Smeekens, S., coding capacity and juxtaposing the 13th amino Rottier, P., and Warren, G. (1984). Sequence acid colon with a stop colon (Schneider et al. and topology of a model intracellular mem- 1994). Following translation of such a minicistron, brane protein, El glycoprotein, from a cor- ribosomes might continue to scan along the RNA onavirus. Nature 308, 751-752. and reinitiate at a downstream AUG colon for the 3) Banerjee, A. K. (1991). Gene expression of nonsegmented negative strand RNA viruses. translation of p57 (from transcripts containing Pharmac. Ther. 51, 47-70. intron-2) or pol (from transcripts lacking intron-2). 4) Bode, L., Riegel, S., Ludwig, H., Amsterdam, J., Removal of intron-2 from the 7.1 kb RNA extends Lange, W. and Koprowski, H. (1988). Borna the pol ORF by 459 nt, increasing the size of the disease virus-specific antibodies in patients putative polymerase protein to 190 kDa. with HIV infection and with mental disorders. Lancet ii, 689. Translation of the 2.7 kb or 7.0 kb RNA to 5) Briese, T., De La Torre, J. C., Lewis, A., synthesize p57 would require that initiation of pro- Ludwig, H., and Lipkin, W. I. (1992). Borna tein synthesis begins at the 4th or 5th AUG colon disease virus, a negative strand RNA virus, from the 5' end. The first AUG colon is known to be transcribes in the nucleus of infected cells. Proc. used for translation of gpl8 while the second and Natl. Acad. Sci. USA 89, 11486-11489. third AUG colons would only result in the synthesis 6) Briese, T., Schneemann, A., Lewis, A., Park, Y., Kim, S., Ludwig, H., and Lipkin, W. I. (1994). of small peptides, 25 and 42 amino acids in length, Genomic organization of Borna disease virus. respectively. Similarly, the sixth AUG colon in the Proc. Natl. Acad. Sci. USA 91, 4362-4366. doubly spliced 6.0 kb mRNA (an AUG colon in a 7) Carbone, K., Duchala, C., Griffin, J., Kincaid, A., strong context) would be used to initiate the synthe- and Narayan, 0. (1987). Pathogenesis of Borna sis of pol. disease in rats. Evidence that intraaxonal spread is the major route for virus dissemina- Summary tion and the determination for disease incuba- tion. J. Virol. 61, 3431-3440. The molecular characteristics of BDV suggest 8) Carbone, K., Park, S. W., Rubin, S. A., Waltrip, that it represents a new taxon within the order R. W., and Vogelsang, G. B. (1991). Borna Mononegavirales. In contrast to other members of disease : association with a maturation defect in the cellular immune response. J. Virol. 65, this order, BDV uses an unprecedented variety of 6154-6164. mechanisms to regulate its gene expression, includ- 9) Caplazi, P., Waldvogel, L., Stitz, L., Braun, U., ing the use of overlapping reading frames, overlap- and Ehrensperger, F. (1994). Borna disease in ping transcription units, alternate RNA splicing and naturally infected cattle. J. Comp. Path. 111, possibly leaky scanning of ribosomes during protein 62-72. translation. Further insights into the mechanistic 10) Castaneda, S. J., and Wong, T. C. (1990). 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