1 Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany
2 Department of Chemical Biology, GBF, Braunschweig, Germany
3 Institut für Virologie, Universität Marburg, Germany
4 Institut für Immunologie, Friedrich Loeffler-Institut, Tübingen, Germany
5 Robert Koch-Institut, D-13353 Berlin, Germany
Correspondence
Martin Schwemmle
martin.schwemmle{at}uniklinik-freiburg.de
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ABSTRACT |
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INTRODUCTION |
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In contrast to other members of the Mononegavirales, BDV replicates in the nucleus of infected cells and uses the splicing machinery for maturation of viral transcripts (Briese et al., 1992; Cubitt et al., 1994
; Schneider et al., 1994
). It encodes at least six viral proteins: the nucleoprotein (N), negative regulator (X), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L) (Briese et al., 1994
). Whereas M and G are involved in particle formation, protein-binding studies suggest that P, N, L and X constitute the polymerase complex. P forms homo-oligomers and can act as a scaffold protein for polymerase complex formation (Schneider et al., 2004
; Schwemmle et al., 1998
; Wolff et al., 2000
). The interaction domain for X is localized to the N-terminal half of P, whereas the oligomerization and L- and N-binding domains are found in the C-terminal half of P (Schneider et al., 2004
; Schwemmle et al., 1998
; Wolff et al., 2000
). Data from a BDV-specific mini-replicon system has challenged the view that X is essential to reconstitute an active polymerase complex, since N, P and L alone were sufficient to achieve replication and transcription (Perez et al., 2003
; Schneider et al., 2003
). Furthermore, additional expression of X resulted in a pronounced inhibition of polymerase activity (Perez et al., 2003
; Schneider et al., 2003
) and thus identified this protein as a negative regulator of the polymerase.
The precise mechanism by which X regulates viral polymerase activity is unclear. It is believed that complex formation between X and P prevents the formation of an active polymerase complex in the nucleus. This hypothesis is based on the observation that co-expression of X and P partially results in cytoplasmic accumulation of both proteins (Kobayashi et al., 2003; Poenisch et al., 2004
). A cytoplasmic localization of XP complexes is also frequently observed in MadinDarby canine kidney (MDCK) cells persistently infected with BDV (Kobayashi et al., 2003
), supporting this hypothesis. Furthermore, P is only found in the nucleus of BDV-infected MDCK cells that lack detectable expression of X (Kobayashi et al., 2003
). However, others found a co-localization of X and P in the nucleus of C6 cells persistently infected with BDV (Schwemmle et al., 1998
), suggesting that the cytoplasmic retention of P by X is most likely not the only mechanism by which X regulates the polymerase activity. Recent observations that P multimers can bind simultaneously to X and L (Schneider et al., 2004
) suggest that ribonucleoprotein (RNP)-bound X could modulate the viral polymerase activity in the nucleus as well.
Data from the BDV mini-replicon assay has revealed that BDV-X can inhibit polymerase activity by 30 % at X : P plasmid ratios of 1 : 6 and almost completely when equimolar amounts are used (Schneider et al., 2003). We therefore hypothesized that the X : P protein ratio is low in persistently infected cells to maintain virus replication and that X is not efficiently incorporated into viral particles, allowing efficient polymerase activity early after infection. To determine unequivocally the subcellular localization and expression levels of X in infected cells, we generated a monoclonal antibody (mAb) against this protein. mAb 10/1G3 specifically recognized a linear epitope of X (70PLHDLRPRP78) and revealed a co-localization of X with P and N in the nucleus and cytoplasm of BDV-infected cells. Based on Western blot analysis, the ratio of X : P : N in crude cell extract was found to be 1 : 6 : 40. Only traces of X were found in concentrated virus stocks, corresponding to an X : P ratio of
1 : 330. Thus, the X protein represents a non-structural protein and cannot interfere with virus replication steps early in infection, whereas the level of X in persistently infected cells is most likely sufficient to exert partial inhibition of the polymerase activity. We propose that X regulates the activity of P directly in the nucleus and not solely by retranslocation into the cytoplasm.
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METHODS |
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To generate an M-specific antibody, New Zealand white rabbits were immunized subcutaneously (s.c.) with 200 µg peptideKLH (M35; N2H-NQFLNIPFLSV-COOH) in complete Freund's adjuvant (Sigma). After 3, 6 and 9 weeks, the rabbits received a second, third and fourth s.c. injection of 200 µg peptideKLH in incomplete Freund's adjuvant (Sigma). Twelve weeks after the initial immunization, blood was taken from the rabbits. The specificity of this antibody was verified by Western blot analysis (data not shown).
Plasmids.
pET15b-based expression plasmids (Novagen) encoding His-tagged BDV N (pHis-N) and P (pHis-P) have been described previously (Schwemmle et al., 1997, 1998
). To generate a His-tagged X expression plasmid (pHis-X), the complete ORF of X (BDV strain He/80) was amplified from plasmid pTRE-X (Schwemmle et al., 1998
) and cloned into the NdeI/BamHI restriction sites of pET15b. In a second step, nucleotide exchanges (49AAT51 to 49GCG51) were introduced by PCR mutagenesis as described by the manufacturer (Invitrogen). The nucleotide exchanges resulted in a single amino acid substitution of the X protein (N17A). The bacterial expression plasmid pMAL-X encoding an MBPX fusion protein was constructed by inserting BDV X from pGEX-p10 (Schneider et al., 2004
; Schwemmle et al., 1998
; Wolff et al., 2000
) cDNA into pMAL-c2 (New England Biolabs).
Protein preparation.
Purification of His-tagged proteins from Escherichia coli was carried out as described previously (Schwemmle et al., 1997). Briefly, cells were suspended in 50 mM Tris/HCl, pH 8·0, 5 mM MgCl2, 500 mM NaCl, 10 % glycerol, 20 mM imidazole and 200 µM Pefabloc (Roth), disrupted by sonication, bound to a Ni-NTA agarose column (Qiagen) and stepwise eluted with 20 mM Tris/HCl, pH 8·0, 5 mM MgCl2, 100 mM NaCl, 10 % glycerol and 500 mM imidazole. Fractions containing His-tagged viral proteins were pooled and stored at 80 °C. The purity of the His-tagged proteins was >95 % as judged by Coomassie blue staining. MBPX fusion protein was purified from bacterial lysates by affinity chromatography on an amylose resin (New England Biolabs) following the instructions of the manufacturer.
Western blot analysis and quantification of protein levels.
BDV-infected or uninfected cells were pooled and lysed in gel loading buffer (Laemmli & Favre, 1973), followed by ultrasonication. Protein extracts were size fractionated by 15 % SDS-PAGE and blotted on to a PVDF membrane (Millipore) for Western blot analysis. The membrane was blocked with milk powder (2 %, w/v, in PBS) for 2 h and then incubated with the indicated antibodies in PBS containing 0·2 % milk powder (w/v) overnight. After intense washing in PBS containing 0·1 % Tween 20 (Sigma), the blot was incubated with a 1 : 2000 dilution of a peroxidase-coupled donkey anti-mouse or anti-rabbit polyclonal antiserum (Dianova) for 1 h at room temperature. Finally, bound enzymic activity was detected using the enhanced chemiluminescence system (ECL+) from Amersham. Signal intensities of the virus-encoded proteins and the known amounts of the E. coli-purified His-tagged marker proteins were determined using ChemiDoc and the software package Quantity One (both from Bio-Rad). Based on these values and the molecular masses of the marker proteins (HisX, 11 421 Da; HisP, 24 479 Da; HisN, 42 985 Da), the amount of virus-encoded protein, as well as the X : P : N ratio, was determined.
Immunofluorescence analysis.
Immunofluorescence analysis was carried out as described previously (Geib et al., 2003) by applying the primary antibodies rabbit anti-N (Geib et al., 2003
), rabbit anti-P (Geib et al., 2003
) and mAb 10/1G3, using a laser scanning microscope (Zeiss).
Peptide array analysis.
Peptide arrays composed of overlapping 15mer fragments with an offset of 3 aa residues representing the protein sequences of X, P, N and M of strain He/80 (Pleschka et al., 2001) were chemically synthesized on cellulose sheets by the spot-synthesis technique as described previously (Frank, 1992
). Probing the arrays with antibodies was essentially carried out as described by Frank (1992)
. Briefly, peptide arrays were blocked with membrane-blocking buffer overnight (Sigma-Genosys), followed by incubation with the corresponding antibody (1 : 1000 dilution) in blocking buffer for 3·5 h at room temperature. After three washes with Tris-buffered saline containing 0·05 % Tween 20, the peptide-bound antibodies were detected by species-specific alkaline phosphatase-conjugated IgG antibodies, which were visualized by the blue-coloured precipitate formed from the BCIP/MTT substrate as described previously (Frank, 1992
). Signal patterns on the membranes were subsequently scanned for documentation.
Virus stock preparation and titration.
Virus stocks were prepared from Oligo cells persistently infected with BDV strain He/80 as described previously (Briese et al., 1992), titrated on Vero cells (Hallensleben et al., 1998
) and concentrated by two steps of ultracentrifugation at 100 000 g for 1 h.
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RESULTS AND DISCUSSION |
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The co-localization of X, P and N in the nucleus of infected cells does not support the hypothesis that X causes a cytoplasmic retention of P (Kobayashi et al., 2003; Poenisch et al., 2004
). Although there is no experimental evidence, an association of X with NP complexes could explain co-localization of these proteins in the nucleus of BDV-infected Oligo cells. The non-overlapping X- and N-binding sites of P (Schneider et al., 2004
; Schwemmle et al., 1998
) may allow the simultaneous interaction with N and X. Alternatively, X may associate with RNP-bound P and thus block the polymerase activity. This is in line with the observation that P multimers can bind to L and X (Schneider et al., 2004
). However, this interaction might only be transient and X may be actively stripped from RNPs prior to packaging. Recently its was shown that the polymerase activity was restored in the presence of X by co-expression of G and M (Perez & de la Torre, 2005
). Thus, G and M might also prevent efficient packaging of X into viral particles.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 June 2005;
accepted 28 July 2005.
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