The Scripps Research Institute, Department of Neuropharmacology IMM-6, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA1
Author for correspondence: Juan Carlos de la Torre. Fax +1 858 784 9981. e-mail juanct{at}scripps.edu
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During the course of studies of the regulation of the synthesis of BDV transcripts initiated at GS3, cytoplasmic RNA (5 µg) isolated from Vero cells uninfected or persistently infected with BDV-He80 was converted into cDNA by priming with the 3' RACE adapter primer (AP) (Gibco BRL) and using Thermoscript reverse transcriptase (RT) at 50 °C for 1 h under conditions recommended by the supplier. PCR with AmpliTaq Gold (Perkin Elmer ABI) and a combination of primer pairs amplified the corresponding cDNA. Analysis of the RTPCR products obtained with the primer pair 2384F and 4724R (Fig. 1C, D
) revealed amplicons with sizes of approximately 2·5 and 1 kb that corresponded to the predicted unspliced and intron II-spliced BDV mRNA species (Cubitt et al., 1994b
; Schneider et al., 1994
). It should be noted that this combination of primers did not allow us to assess the presence or absence of intron I sequences. RNA from BDV-infected cells also yielded an unexpected amplicon of about 200 bp. Similar results were obtained in cells transfected with plasmid pCAGSpG/L, which contains nt 22368819 of the BDV-HE80 genome, spanning from the AUG of the G ORF to the stop codon of the L ORF (Fig. 1D
). To determine the sequence relationship between the PCR products obtained and the BDV genome, we cloned and sequenced the 1 kb and 200 bp PCR products. This analysis verified that the 1 kb amplicon was derived from previously characterized intron II-spliced BDV mRNA species. The 200 bp PCR product lacked the region between nt 2410 and 4559 of the BDV genome (deletion III). Inspection of the BDV sequences (antigenomic polarity) at the boundaries of deletion III revealed the presence of sequence motifs similar to the consensus splice site elements for a typical metazoan intron (Green, 1986
). The upstream breaking point corresponded to a previously reported splice donor site (SD2) (Fig. 1B
). Sequences characteristic of 3' splice sites, namely the branch site region, polypyrimidine tract and AG dinucleotide, preceded the 3' end of the break point (Fig. 1E
). Therefore, deletion III was considered to be a new intron, intron III, in the BDV genome. Splicing of intron III uses the previously described SD2 and an alternative 3' splice site (SA3) (Fig. 1E
). Consistent with other reports (Jehle et al., 2000
), authentic BDV pre-mRNA was spliced with significantly lower efficiency than cDNA-derived viral pre-mRNA (Fig. 1D
).
Two new ORFs with products of estimated molecular masses of 8·4 (p8·4) and 165 (p165) kDa are predicted in intron III-spliced BDV mRNAs (Fig. 2A). The stop codon of ORF p8·4 was in close proximity to GE5*, a signal that was thought not to operate in virus-infected cells. It seemed plausible, however, that the use of GE5* could facilitate expression of p8·4. Therefore, we revisited the question of whether GE5* is active in BDV-infected cells. For this purpose, cDNA generated by priming RNA from virus-infected cells with the 3' RACE AP primer was subjected to PCR with primers 2384F and the universal adapter primer (UAP) (Gibco BRL). The UAP primer is complementary to specific sequences present in the 3' RACE AP primer. A fragment of about 275 bp was amplified from RNA from infected cells, but not from uninfected controls (not shown). Cloning and sequencing of this PCR product verified the presence at its 3' end (BDV antigenomic polarity) of GE5*. These findings indicated that GE5* is used in BDV-infected cells. The predicted products of ORFs p8·3 and p165 will contain the signal peptide sequence of BDV G. Hence, p8·3 and p165 proteins could be targetted to the ER and enter the secretory pathway (Doms et al., 1993
). Consistent with this hypothesis, plasmid-derived p165 protein tagged with a c-Myc epitope accumulated in the ER/Golgi apparatus (Fig. 2B
). Expression of p8·3 and p165 proteins, and their possible secretion to the extracellular milieu in virus-infected cells, remains to be determined. However, it is worth noting the presence of three RGD motifs in p165, which could provide a secreted p165 protein with the ability to interact with integrin molecules present at the cell surface (Ruoslahti & Pierschbacher, 1987
). This, in turn, could trigger cellular signal transduction pathways that might contribute to BDVcell interactions. These findings suggest that alternative splicing of BDV pre-mRNA may generate a variant L gene product with functions other than those predicted for an RNA-dependent RNA polymerase. A similar situation has been proposed for the cytomegalovirus DNA polymerase accessory protein, ppM44 (Loh et al., 2000
).
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Our findings provide additional evidence that, among the known mononegaviruses, BDV exhibits unique features with respect to the regulation of its genome expression. Consistent with a previous report (Jehle et al., 2000 ), we also observed an apparent lower efficiency of splicing in virus-derived BDV mRNAs compared with plasmid-derived BDV mRNAs. In addition, we observed a similar splicing pattern in different cell types from different species that was not altered significantly in response to osmotic shock-mediated stress. These results suggest that BDV might have developed strategies that provide it with some degree of insulation from cellular influences that could have unwanted effects on the regulation of virus RNA processing. The same mechanisms may also prevent BDV-induced disturbances of the regulation of the cellular RNA processing machinery, thus facilitating virus persistence without compromising cell viability. The elucidation of the mechanisms underlying the regulation of RNA splicing during BDV RNA synthesis, as well as the identification and functional characterization of the new BDV polypeptides predicted from intron III-spliced viral mRNAs in BDV-infected cells, are expected to contribute significantly to a better understanding of the biology of BDV.
While this manuscript was under review, Tomonaga et al. (2000) published findings similar to those presented here. In addition, these authors also identified a negative regulatory activity of SA3 splicing in the BDV genome (Tomonaga et al., 2000
).
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References |
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Berget, S. M.(1995). Exon recognition in vertebrate splicing. Journal of Biological Chemistry 270, 2411-2414.
Briese, T., de la Torre, J. C., Lewis, A., Ludwig, H. & Lipkin, W. I.(1992). Borna disease virus, a negative-strand RNA virus, transcribes in the nucleus of infected cells. Proceedings of the National Academy of Sciences, USA 89, 11486-11489.[Abstract]
Briese, T., Schneemann, A., Lewis, A. J., Park, Y. S., Kim, S., Ludwig, H. & Lipkin, W. I.(1994). Genomic organization of Borna disease virus. Proceedings of the National Academy of Sciences, USA 91, 4362-4366.[Abstract]
Canman, C. E. & Kastan, M. B.(1996). Signal transduction. Three paths to stress relief. Nature 384, 213-214.[Medline]
Carbone, K. M., Rubin, S. A., Sierra-Honigmann, A. M. & Lederman, H. M.(1993). Characterization of a glial cell line persistently infected with Borna disease virus (BDV): influence of neurotropic factors on BDV protein and RNA expression. Journal of Virology 67, 1453-1460.[Abstract]
Chabot, B.(1996). Directing alternative splicing: cast and scenarios. Trends in Genetics 12, 472-478.[Medline]
Cubitt, B. & de la Torre, J. C.(1994). Borna disease virus (BDV), a nonsegmented RNA virus, replicates in the nuclei of infected cells where infectious BDV ribonucleoproteins are present. Journal of Virology 68, 1371-1381.[Abstract]
Cubitt, B., Oldstone, C. & de la Torre, J. C.(1994a). Sequence and genome organization of Borna disease virus. Journal of Virology 68, 1382-1396.[Abstract]
Cubitt, B., Oldstone, C., Valcarcel, J. & de la Torre, J. C.(1994b). RNA splicing contributes to the generation of mature mRNAs of Borna disease virus, a non-segmented negative strand RNA virus. Virus Research 34, 69-79.[Medline]
de la Torre, J. C.(1994). Molecular biology of Borna disease virus: prototype of a new group of animal viruses. Journal of Virology 68, 7669-7675.[Medline]
Doms, R. W., Lamb, R. A., Rose, J. K. & Helenius, A.(1993). Folding and assembly of viral membrane proteins. Virology 193, 545-562.[Medline]
Green, M. R.(1986). Pre-mRNA splicing. Annual Review of Genetics 20, 671-708.[Medline]
Jehle, C., Lipkin, W. I., Staeheli, P., Marion, R. M. & Schwemmle, M.(2000). Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs. Journal of General Virology 81, 1947-1954.
Kanopka, A., Muhlemann, O., Petersen-Mahrt, S., Estmer, C., Ohrmalm, C. & Akusjarvi, G.(1998). Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins. Nature 393, 185-187.[Medline]
Kyriakis, J. M. & Avruch, J.(1996). Sounding the alarm: protein kinase cascades activated by stress and inflammation. Journal of Biological Chemistry 271, 24313-24316.
Lamb, R. A. & Horvath, C. M.(1991). Diversity of coding strategies in influenza viruses. Trends in Genetics 7, 261-266.[Medline]
Loh, L. C., Locke, D., Melnychuk, R. & Lafertet, S.(2000). The RGD sequence in the cytomegalovirus DNA polymerase accessory protein can mediate cell adhesion. Virology 272, 302-314.[Medline]
Lopez, A. J.(1998). Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annual Review of Genetics 32, 279-305.[Medline]
Pyper, J. M. & Clements, J. E.(1994). Partial purification and characterization of Borna disease virions released from infected neuroblastoma cells. Virology 201, 380-382.[Medline]
Ruoslahti, E. & Pierschbacher, M. D.(1987). New perspectives in cell adhesion: RGD and integrins. Science 238, 491-497.[Medline]
Sauder, C., Muller, A., Cubitt, B., Mayer, J., Steinmetz, J., Trabert, W., Ziegler, B., Wanke, K., Mueller-Lantzsch, N., de la Torre, J. C. & Grasser, F. A.(1996). Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. Journal of Virology 70, 7713-7724.[Abstract]
Schneemann, A., Schneider, P. A., Kim, S. & Lipkin, W. I.(1994). Identification of signal sequences that control transcription of Borna disease virus, a nonsegmented, negative-strand RNA virus. Journal of Virology 68, 6514-6522.[Abstract]
Schneemann, A., Schneider, P. A., Lamb, R. A. & Lipkin, W. I.(1995). The remarkable coding strategy of Borna disease virus: a new member of the nonsegmented negative strand RNA viruses. Virology 210, 1-8.[Medline]
Schneider, P. A., Schneemann, A. & Lipkin, W. I.(1994). RNA splicing in Borna disease virus, a nonsegmented, negative-strand RNA virus. Journal of Virology 68, 5007-5012.[Abstract]
Schneider, P. A., Kim, R. & Lipkin, W. I.(1997). Evidence for translation of the Borna disease virus G protein by leaky ribosomal scanning and ribosomal reinitiation. Journal of Virology 71, 5614-5619.[Abstract]
Smith, C. W. J. & Valcárcel, J.(2000). Alternative pre-mRNA splicing: the logic of combinatorial control. Trends in Biochemical Sciences 25, 381-388.[Medline]
Tomonaga, K., Kobayashi, T., Lee, B.-J., Watanabe, M., Kamitani, W. & Ikuta, K.(2000). Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus. Proceedings of the National Academy of Sciences, USA 97, 12788-12793.
van der Houven van Oordt, W., Diaz-Meco, M. T., Lozano, J., Krainer, A. R., Moscat, J. & Caceres, J. F.(2000). The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. Journal of Cell Biology 149, 307-316.
Walker, M. P., Jordan, I., Briese, T., Fischer, N. & Lipkin, W. I.(2000). Expression and characterization of the Borna disease virus polymerase. Journal of Virology 74, 4425-4428.
Received 21 September 2000;
accepted 24 November 2000.