Department of Virologie, Institute for Medical Microbiology & Hygiene, University of Freiburg, Hermann-Herder Str. 11, D-79104 Freiburg, Germany1
Emerging Diseases Laboratory, Departments of Neurology, Anatomy and Neurobiology, and Microbiology and Molecular Genetics, University of California-Irvine, Irvine, CA 92697-4292, USA2
Centro Nacional de Biotecnologia (CSIC), Cantoblanco, 28049 Madrid, Spain3
Author for correspondence: Martin Schwemmle. Fax +49 761 203 6639. e-mail schwemm{at}ukl.uni-freiburg.de
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Abstract |
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Introduction |
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Unlike other non-segmented negative-strand RNA viruses, Borna disease virus (BDV) replicates and transcribes the genome in the nucleus (Briese et al., 1992 ; Cubitt & de la Torre, 1994
). Four major subgenomic RNAs (1·2, 0·8, 2·8 and 7·1 kb RNA) are transcribed from three transcription units of the BDV genome (Briese et al., 1994
; Schneemann et al., 1994
). Whereas the 1·2 kb RNA, encoding the nucleoprotein (N), is monocistronic, all the other RNAs are multicistronic. The 0·8 kb RNA encodes the phosphoprotein (P) and the X-Protein. The 2·8 kb RNA contains the ORFs for the putative matrix protein (M) and the glycoprotein (G). The 7·1 kb RNA encodes the polymerase (L) as well as M and G (Briese et al., 1994
). The 2·8 and 7·1 kb RNAs are co-terminal at the 5' end but differ in length due to transcriptional readthrough at a termination signal (Schneemann et al., 1994
). Since only about 3% readthrough is observed at this termination signal, the 2·8 kb RNA represents the major transcript of the third transcription unit (Schneemann et al., 1994
). Expression of M and G from the 2·8 kb RNA, and L from 7·1 kb RNA, are regulated by splicing (Cubitt et al., 1994b
; Schneider et al., 1994
). Whereas intron 1 is located within the M-ORF, intron 2 is found within the ORFs for G and L. Therefore, transcripts retaining intron 1 serve primarily as mRNAs for M. Splicing of intron 1 results in the disruption of the M-ORF, leaving a minicistron encoding only eight amino acids, which enhances expression of G (Schneider et al., 1997a
). Splicing of intron 2 creates the functional L-ORF in the 7·1 kb RNA (Schneider et al., 1994
). Additional splicing of intron 1 is most likely required to facilitate initiation at the AUG of the L-ORF. Thus, differential splicing of both introns is critical for balanced expression of the viral proteins M, G and L.
Little is known about the cytoplasmic levels of the various splice forms of BDV RNAs (Cubitt et al., 1994b ; Schneider et al., 1994
, 1997b
). It is unclear whether virus-specific factors/elements are involved in the regulation of splicing. First indications that such factors might be implicated emerged in a recent study where a cis-acting RNA element located within the 3' non-coding region of the 2·8 kb RNA was found to be important for efficient cytoplasmic accumulation of unspliced RNA (Schneider et al., 1997b
).
We have examined levels of unspliced and spliced BDV transcripts in infected cells and cells transfected with expression constructs encoding the 2·8 kb RNA. BDV-encoded transcripts were spliced less efficiently than those encoded by expression constructs. The splicing efficiency of transcripts generated from expression constructs was similar in uninfected and infected cells. These findings suggest that splicing of BDV-encoded transcripts may be co-transcriptionally regulated.
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Methods |
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Construction of plasmids.
Plasmids pRPAwt and pRPAmt were used for in vitro transcription of the wild-type RNase protection assay (RPA) probe (RPAwt) or the mutant RPA probe (RPAmt) and were generated as follows: To create pRPAwt, the HindIIIEcoRI fragment of pRPA-1 (Schneider et al., 1997b ), encoding part of the BDV M/G-ORFs corresponding to nucleotides 19752510 of BDV strain He/80 (Cubitt et al., 1994a
), was cloned into pGEM-4 (Promega). Plasmid pRPAmt was derived from pRPAwt by replacing six nucleotides of exon 2 (ccagag to gttaac) of the M/G-ORFs corresponding to nucleotides 22162221 of BDV strain He/80 using the Quick-mutagenesis protocol (Roche Biochemicals). To clone the mutated DNA into plasmid pRc2.8 encoding the 2·8 kb RNA of BDV (Schneider et al., 1997b
), the BbsIBst1107I fragment of pRPAmt, harbouring the respective region, was first subcloned into plasmid pBS-XbaI containing the XbaI fragment of pRc2.8, resulting in plasmid pBS-XbaI/mt. In a final step, the XbaI fragment of pRc2.8 was replaced with the corresponding fragment of pBS-XbaI/mt, resulting in plasmid pRc2.8mt. Plasmids pRc2.6 and pRc1.5 were obtained by ligating NotI fragments of plasmids p2.6 and p1.5 (kindly provided by Patrick Schneider, UC-Irvine, USA) encoding either the 2·6 kb RNA or 1·5 kb RNA of BDV strain He/80 into the corresponding sites of pRc2.8. Plasmid pG3T.t was used to generate the RPA probe for detection of SV40 t-antigen mRNAs in COS-7 cells. Plasmid pG3T.t was prepared by cloning a PCR product including the sequences from position 4541 to 4978 in SV40 DNA into pGEM-3 (Promega). The primers used for amplification (5' gcgcgcgaattcggaagatggagtaaaatatgc and 5' gcgcgcggatccgctcccattcatcagttcc) contained additional EcoRI and BamHI sites. The PCR product was digested with both enzymes and cloned into EcoRI-and BamHI-digested pGEM-3 DNA. Plasmids encoding the N-ORF (pCMV-N) and X-ORF (pCMV-X) of BDV strain He/80 under control of a CMV promoter were kindly provided by Jürgen Hausmann, University of Freiburg, Germany.
RNA preparation.
Isolation of poly(A)+ RNA from total RNA was carried out using the Oligotex mRNA spin column protocol from Qiagen. RNA from cytoplasmic and nuclear fractions were obtained as described (Nevins, 1980 ). Briefly, cells were trypsinized 48 h post-transfection, harvested and washed twice with PBS. Cell pellets were lysed in 500 µl Iso-Hi-pH containing 10 mM TrisHCl (pH 8·4), 140 mM NaCl, 1·5 mM MgCl2 and 0·5% Nonidet P-40 (0·5%, v/v) for 5 min at 4 °C. Nuclear and cytoplasmic fractions were subsequently separated by centrifugation at 1000 g for 3 min at room temperature. The crude nuclear pellet was immediately washed once in 500 µl Iso-Hi-pH and once in 500 µl Iso-Hi-pH containing 0·33% (w/w) sodium deoxycholate and 0·66% (v/v) Tween 40 to obtain the final nuclear fraction. Total RNA and RNA from both cytoplasmic and nuclear fractions was prepared with peqGOLD TriFast (Peqlab) as recommended by the manufacturer.
In vitro transcription and RNase protection assay.
Run-off transcriptions were performed at 37 °C in a total volume of 20 µl containing 0·2 µg linearized DNA, 1xT7 transcription buffer (Roche Biochemicals), 28 U RNasin (Amersham), 0·5 µM ATP, GTP and CTP, 0·05 µM UTP (Pharmacia), 3 U T7 Polymerase (MBI Fermentas) and 1 µCi 32P-labelled rUTP (3000 Ci/mmol; Amersham) for 90 min. For subsequent DNase digestion the total volume was raised to a final volume of 30 µl with 1xtranscription buffer including 4 U RNase-free DNase (Ambion) and incubated for 30 min at 37 °C. Purification of the 32P-labelled RNA and subsequent RPA reactions was done as described in Stalder et al. (1998) . RPA was performed with approximately 1·4x105 c.p.m. 32P-labelled RPA-probe and either 5 µg of total RNA or RNA from the cytoplasmic fraction, 1 µg of RNA from the nuclear fraction or poly(A)+ RNA, prepared from 50 µg of total RNA. Digestion of unhybridized RNA was performed using 0·02 µg per sample RNase A (Ambion) and 16 U per sample RNase T1 (Roche Biochemicals). The RPA signals were quantified with a phosphorimager (FUJIX BAS1000) using the software package MacBAS version 2.2 (Fuji). Ratios of unspliced and spliced RNA species of one RPA reaction were calculated based on the signal intensities and were normalized to the number of U residues in the respective protected probe fragments to account for the bias of signal intensities caused by the different lengths of the RNA fragments. RPA size markers were obtained by PCR using pRc2.8 and primers C1 (5' ttgtaggagggacttcacgg), C2 (5' ctgtagctaccaaaggatcc), C3 (5' tgaaagagcctctgcag) and C4 (5' ctcgcatgaaatgacattt), resulting in four DNA fragments of length 535, 484, 434 and 383 bp, respectively. These amplification products (20 ng per fragment) were used in the RPA reaction to generate fragments of the radiolabelled RPA probe corresponding to the length of the expected unspliced and spliced viral RNA species.
Virus stock preparation and titration.
Virus stock from OL cells persistently infected with BDV strain He/80 (Cubitt et al., 1994a ) was prepared as described in Briese et al. (1992)
with slight modifications. Briefly, 25 confluent 90 mm plates cells were washed with 20 mM HEPES (pH 7·4) and incubated with 10 ml 20 mM HEPES (pH 7·4) containing 250 mM MgCl2 and 1% FCS for 1·5 h at 37 °C. Subsequently, supernatants were harvested and centrifuged twice at 2500 g for 5 min to remove cell debris. Virus particles were concentrated by ultracentrifugation for 1 h at 20 °C at 80000 g onto a 20% sucrose cushion containing 20 mM HEPES (pH 7·4) and 1% FCS. Virus-containing pellets were resuspended in PBS to approximately 106 f.f.u./ml. Persistent BDV infection was established by an initial infection of 105 C6 , Vero , MDCK or COS-7 cells with 104 f.f.u. of virus stock followed by at least 5 weeks of passaging. Complete infection of the cultures was monitored by immunofluorescence assay as described by Hallensleben & Staeheli (1999)
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Results |
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Splicing of plasmid-encoded 2·8 kb RNA differs from that of virus-encoded transcripts
To test the hypothesis that the ratio of spliced and unspliced RNAs in BDV-infected cells is controlled by intrinsic features of the viral 2·8 kb or 7·1 kb RNA we transfected plasmid pRc2.8, which expresses only the 2·8 kb RNA, into COS-7 cells (Fig. 2). Two days after transfection we prepared RNA from total cell lysates or from cytoplasmic and nuclear extracts, and analysed the RNA samples for the presence of the various splice forms using the RPA probe described above. As residual plasmid DNA in the RNA preparations would provide signals indistinguishable from unspliced 2·8 kb RNA, parallel RNA samples were treated with RNase-free DNase prior to the RPA. The importance of this control is illustrated by the presence of a strong signal at the position of unspliced transcripts shown in Fig. 2
, particularly in the total and the nuclear RNA fraction (lanes 6 and 8); this signal was indeed substantially reduced in DNase-treated samples (lanes 9 and 11). All four splice forms were detected in total RNA and the cytoplasmic fraction (Fig. 2
, lanes 6, 7, 9 and 10). Transcripts originating from 2·8 kb RNA expression plasmids were preferentially intron 1-spliced and double-spliced (Fig. 2
, lane 10). In contrast, higher levels of unspliced transcripts were observed in BDV-infected COS-7 cells (Fig. 2
, lane 5). Cytoplasmic concentrations of unspliced and intron 2-spliced transcripts were 9 and 4%, respectively, as compared to 47 and 28% in BDV-infected COS-7 cells (Table 2
).
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Splicing of cDNA-derived 2·8 kb RNA remains unchanged in the presence of viral proteins
The differences in the splice ratios of cDNA-derived 2·8 kb RNA and virus-encoded RNAs could be explained by viral factors. Pilot experiments indicated that co-transfection of plasmids encoding the viral proteins N, P or X either alone or in combination into Vero cells expressing the viral 2·8 kb RNA, did not influence splicing of intron 1 and intron 2 (data not shown). To address the possibility that other transacting viral factors might be missing, we studied splicing of the 2·8 kb RNA in BDV-infected COS-7 cells. To facilitate discrimination between virus and plasmid-encoded RNAs we used a plasmid (pRc2.8mt) that coded for a mutant form of the 2·8 kb RNA carrying six nucleotide exchanges in exon 2. RNA samples were analysed 48 h post-transfection by RPA using RPAmt as a probe, which is complementary to mutated 2·8 kb RNA. Since virus-encoded RNAs do not perfectly match RPAmt, the RNases cleave the probe into two fragments of smaller size. As shown in Fig. 3, expression of the mutated 2·8 kb RNA revealed a splice pattern in BDV-infected COS-7 cells (lane 18) similar to that observed in uninfected COS-7 cells (lane 16). This pattern was identical to that seen in COS-7 cells transfected with pRc2.8, which encodes the wild-type form of the 2·8 kb RNA (lane 6). Comparable splice ratios were observed in uninfected and BDV-infected COS-7 cells using different amounts of pRc2.8mt per transfection (ranging from 0·1 to 1 µg; data not shown). Thus, BDV infection did not seem to influence splicing of cDNA-derived 2·8 kb RNA. However, expression of the mutant form of the 2·8 kb RNA resulted in a subtle change in the splice pattern of virus-encoded RNAs (compare Fig. 3
, lane 5 and 9): the amounts of intron 1-spliced and double-spliced transcripts seemed to increase under these conditions.
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Discussion |
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Attenuation of transcription at the gene borders is a feature of nonsegmented negative-strand RNA viruses that results in a concentration gradient of mRNAs encoding sequential ORFs (Conzelmann, 1998 ). The pattern of gene expression is more complex in BDV because M and G are derived from the multicistronic 2·8 and 7·1 kb RNAs. The unspliced and intron 2-spliced mRNAs that encode M are more abundant in the cytoplasm of BDV-infected cells than the intron 1-spliced mRNAs that encode G. Thus, BDV establishes a concentration gradient similar to that observed in other Mononegavirales at least in part by regulating the splicing of intron 1 and intron 2. A comparison with plasmid-encoded 2·8 kb RNA indicates that splicing in BDV-infected cells is not solely due to intrinsic features of the viral RNA. We therefore speculate that BDV-encoded factors may interfere with splicing of both introns. This could be achieved by promoting nuclear export of unspliced and partially spliced mRNAs. Intrinsically inefficient splicing of intron 2 might cause the preferential cytoplasmic accumulation of intron 1-spliced RNA. This view is supported by our observation that plasmid-encoded splice intermediates lacking intron 1 were not spliced as efficiently as those intermediates lacking intron 2. However, recent observations indicate that transcription and processing of the cellular polymerase II transcripts is tightly linked, most likely due to the spatial relationship of the different enzymes involved (McCracken et al., 1997
). Therefore, an alternative explanation for the preferential splicing of the plasmid-derived transcripts might be that these transcripts are generated in an environment more active in RNA processing than the compartment in which BDV transcription occurs.
In this study we were unable to confirm the results of our earlier report wherein the unspliced plasmid-encoded 2·8 kb RNA predominantly accumulated in the cytoplasm due to an RNA element located in the 3' non-coding region (Schneider et al., 1997b ). Since we used the same detection technique (RPA), plasmid (pRc2.8) and cell type (COS-7 cells), we propose that the differences may relate to the fact that the RNA samples in this study were subjected to DNase treatment before analysis by RPA. Furthermore, expression of 2·8 kb RNA lacking the putative export sequence did not result in an enhanced cytoplasmic accumulation of spliced RNA (C. Jehle, N. Horscroft, W. I. Lipkin & M. Schwemmle, unpublished results), indicating that regulation of viral RNA splicing in infected cells most likely occurs via other mechanisms and not as proposed through the 3' non-coding region (Schneider et al., 1997b
). One alternative is that nuclear export of unspliced BDV transcripts may be coupled to transcription by the viral polymerase. Support for this hypothesis was found in the observation that the cytoplasmic levels of unspliced 2·8 kb RNA originating from transfected plasmid DNA were low in both infected and uninfected COS-7 cells (Fig. 3
, lanes 16 and 18). Intriguingly, we detected subtle changes in virus-encoded RNA species when large amounts of 2·8 kb RNA were expressed from transfected plasmids (Fig. 3
, lane 9), suggesting interference with viral factors required for regulation of splicing.
BDV infection does not appear to interfere with the processing of cellular intron-containing RNA polymerase II transcripts including t-antigen pre-mRNAs in COS-7 cells (Fig. 4) or trKc pre-mRNAs (Valenzuela et al., 1993
) in brains of newborn rats (C. Sauder, personal communication). Regulation of differential splicing at the co-transcriptional level provides a mechanism for control of BDV gene expression that does not compromise the viability of the host cell or the potential for establishment of persistent infection.
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Acknowledgments |
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References |
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Received 22 December 1999;
accepted 4 April 2000.