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Short Communication |
Department of Virology, University of Freiburg, D-79104 Freiburg, Germany
Correspondence
Peter Staeheli
peter.staeheli{at}uniklinik-freiburg.de
Urs Schneider
urs.schneider{at}uniklinik-freiburg.de
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ABSTRACT |
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These authors contributed equally to this work. 
Pictures of brain sections from infected mice stained with a mAb directed against the BDV nucleoprotein are available with the online version of this paper.
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) which belongs to the order Mononegavirales (de la Torre, 1994; Schneemann et al., 1995). BDV naturally infects the central nervous system (CNS) of a wide range of mammalian species and is the causative agent of Borna disease (Rott & Becht, 1995), an immune-mediated neurological disorder mainly affecting horses and sheep (Ludwig et al., 1988). Successful experimental infection of a variety of warm-blooded animals has been reported (Rott & Becht, 1995; Staeheli et al., 2000). Nevertheless, most tissue culture-adapted laboratory strains of BDV do not readily infect the CNS of mice, but can be adapted to mice by serial passage. Recently, we successfully adapted molecularly cloned BDV to the mouse and showed that adaptive mutations in the L polymerase (L1116R and N1398D) as well as in the polymerase cofactor P (R66K) contributed to replication competence of BDV in mice (Ackermann et al., 2007). In a previous study, Nishino et al. (2002) identified four different amino acid changes in a derivative of BDV strain He/80, designated CRNP5, that unlike its parent propagates efficiently in the brain of mice. Other amino acid changes originally reported to be present in CRNP5 (Nishino et al., 2002) could not be confirmed if the sequence was compared with those of BDV strain He/80FR (data not shown). The remaining amino acid changes in CRNP5 affected the viral glycoprotein G (F458S and Y480H) and the polymerase L (K1417R and G1686R).
To determine the relative contributions of these four mutations, we used reverse genetics technology established by our group (Martin et al., 2006; Schneider et al., 2005). We generated full-length cDNAs carrying either the amino acid substitutions F458S and Y480H in G, the substitutions K1417R and G1686R in L or all four substitutions in combination and recovered the corresponding viruses. The resulting recombinant viruses were designated BDV-GSH, BDV-LRR and BDV-GSH/LRR, respectively (Fig. 1). To further analyse the individual contributions of the mutations at amino acid positions 1417 and 1686 in L, we created full-length cDNAs carrying these mutations individually. The corresponding viruses were recovered and were designated BDV-L1417R and BDV-L1686R, respectively (Fig. 1).
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2-microglobulin-deficient MRL mice with 1000 focus-forming units (f.f.u.) of each mutant virus by the intracerebral route. These mice are highly susceptible to infection as they lack mature CD8 T cells which represent the main antiviral defence system against BDV (Hallensleben et al., 1998). The animals were killed 8 weeks post-infection and analyzed for the presence of BDV in the brain. One brain hemisphere of each animal was embedded in paraffin and stained for the BDV nucleoprotein N as described previously (Rauer et al., 2004). The second brain hemisphere was used to determine the content of BDV-specific RNA by quantitative RT-PCR in which an amplicon (primers 5'-CATGGTGAGACTGCTACAC-3' and 5'-CTCAAAGTCTGTAGTTAGTAG-3', and probe 5'-6FAM-ATCCAATCTATAGCCTCATGTGGT-TAMRA-3') specific for the BDV-N gene was employed. One hundred nanograms of total RNA derived from a complete brain hemisphere was reverse transcribed and amplified by one-step RT-PCR using the Light Cycler RNA master hybridization probe kit from Roche. The specificity of the amplicon was verified using an in vitro-transcribed RNA representing the BDV N gene (data not shown). As expected, CRNP5 grew extremely well in all five infected mice, whereas the non-adapted parental virus grew very poorly (Fig. 2). All four mice infected with BDV-GSH/LRR contained a high number of infected cells in the brain (Fig. 2a). Viral RNA levels in brains of animals infected with BDV-GSH/LRR were only slightly lower than those of animals infected with CRNP5 (Fig. 2b). BDV-LRR that carries the two polymerase mutations, but not the glycoprotein mutations, grew almost as efficiently in the mouse brains as BDV-GSH/LRR, indicating that the polymerase mutations mainly conferred replication competence in mice. The poor growth of single mutants BDV-L1417R and BDV-L1686R (Fig. 2) further demonstrated that the simultaneous presence of both L mutations was required for efficient growth of BDV in the mouse CNS.
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) and viral nucleic acids (Fig. 2b). One possible explanation of this result was that BDV-GSH tended to acquire additional mutations which facilitate virus replication in the mouse brain. To evaluate this possibility, we sequenced overlapping RT-PCR fragments of the complete coding sequence of the virus that grew best under these conditions (arrow in Fig. 2b). Sequence analysis confirmed the presence of the two intentionally introduced mutations in G, but also revealed the presence of a single additional mutation in the L gene at position 7034 of the BDV antigenome. The newly acquired mutation changed leucine at position 1116 in the L polymerase to arginine. Interestingly, we previously identified this particular amino acid change in L as a critical alteration that confers the replication competence of molecularly cloned He/80 in mouse brains (Ackermann et al., 2007). Sequencing of the critical L gene region of BDV-GSH from four additional mice with high or intermediate virus load in the brain showed the same amino acid substitution, indicating that the G mutations greatly favour the selection of additional adaptive mutations in L.
How could mutations in L enhance virus replication in mice? One explanation was that amino acid substitutions L1417R and G1686R enhanced polymerase activity. BDV polymerase activity can be measured by reconstituting the viral polymerase complex in transfected cells by simultaneous expression of artificial viral genomes encoding a reporter gene and the viral proteins N, P and L (Perez et al., 2003; Schneider et al., 2003). If human 293T cells were used for such transfection experiments, the activity of polymerase complexes containing mutant LRR was not significantly increased compared with the activity of the wild-type complex (Fig. 3a). In contrast, if BSR-T7 hamster cells (Fig. 3b) and neuronal mouse N2A cells (Fig. 3c) were transfected, we detected a statistically significant increase in the activity of polymerase complexes reconstituted with mutant LRR which was 3.5- and-8 fold higher, respectively, than the activity of complexes containing wild-type L (Fig. 3b, c). These results indicated that the mutant polymerase exhibits enhanced activity in rodent cells.
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). One of the identified adaptive mutations resulted in reduced negative regulation of the viral polymerase complex by the viral X protein, while the other mutations apparently did not change the activity of the viral polymerase complex as determined in minireplicon assays. The decisive mutation in L was identical to that which newly appeared during the growth of BDV-GSH in the course of this study. The ability of BDV-GSH to acquire this additional mutation in infected mice suggests a substantial contribution of improved cell-to-cell spread in the adaptation process of BDV to a new host species. Nevertheless, our results strongly indicate that the viral polymerase complex is the main determinant of the BDV host range and that properly tuned polymerase activity can permit replication in cells which are usually unsuitable substrates for BDV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Briese, T., Schneemann, A., Lewis, A. J., Park, Y. S., Kim, S., Ludwig, H. & Lipkin, W. I. (1994). Genomic organization of Borna disease virus. Proc Natl Acad Sci U S A 91, 4362–4366.
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Hallensleben, W., Schwemmle, M., Hausmann, J., Stitz, L., Volk, B., Pagenstecher, A. & Staeheli, P. (1998). Borna disease virus-induced neurological disorder in mice: infection of neonates results in immunopathology. J Virol 72, 4379–4386.
Ludwig, H., Bode, L. & Gosztonyi, G. (1988). Borna disease: a persistent virus infection of the central nervous system. Prog Med Virol 35, 107–151.[Medline]
Martin, A., Staeheli, P. & Schneider, U. (2006). RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication. J Virol 80, 5708–5715.
Nishino, Y., Kobasa, D., Rubin, S. A., Pletnikov, M. V. & Carbone, K. M. (2002). Enhanced neurovirulence of Borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes. J Virol 76, 8650–8658.
Perez, M., Sanchez, A., Cubitt, B., Rosario, D. & de la Torre, J. C. (2003). A reverse genetics system for Borna disease virus. J Gen Virol 84, 3099–3104.
Rauer, M., Gotz, J., Schuppli, D., Staeheli, P. & Hausmann, J. (2004). Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease. J Virol 78, 3621–3632.
Rott, R. & Becht, H. (1995). Natural and experimental Borna disease in animals. Curr Top Microbiol Immunol 190, 17–30.[Medline]
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.[CrossRef][Medline]
Schneider, U., Naegele, M., Staeheli, P. & Schwemmle, M. (2003). Active Borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein. J Virol 77, 11781–11789.
Schneider, U., Schwemmle, M. & Staeheli, P. (2005). Genome trimming: a unique strategy for replication control employed by Borna disease virus. Proc Natl Acad Sci U S A 102, 3441–3446.
Staeheli, P., Sauder, C., Hausmann, J., Ehrensperger, F. & Schwemmle, M. (2000). Epidemiology of Borna disease virus. J Gen Virol 81, 2123–2135.
Received 16 May 2007;
accepted 23 July 2007.
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