Institut für Virologie, Philipps-Universität Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany1
Institut für Virologie, Justus Liebig Universität Giessen, Frankfurter Str. 107, 35392 Giessen, Germany2
Author for correspondence: Thorsten Wolff. Present address: Robert-Koch-Institut, Nordufer 20, 13353 Berlin, Germany. Fax +49 30 4547 2328. e-mail wolfft{at}rki.de
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Abstract |
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Introduction |
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The genome organization of BDV is similar to that of related members of the order Mononegavirales (de la Torre, 1994 ; Schneemann et al., 1995
). Initially, five large open reading frames were identified and it was suggested that they encoded BDV-specific proteins that function as a nucleoprotein (p38/p39), phosphoprotein (p24), matrix protein (gp18), surface glycoprotein (gp94) and RNA-dependent RNA polymerase (p180/190). We have recently demonstrated that BDV expresses a sixth gene product of 10 kDa, the p10 protein, that is encoded by the predicted x1 open reading frame (Cubitt et al., 1994
) which initiates upstream of and partially overlaps with the p24 coding region (Wehner et al., 1997
). The p10 protein has no significant sequence homology to any known protein. However, p10 was found to co-localize with the viral nucleoprotein in the nucleus of BDV-infected cells suggesting an association with the viral ribonucleoprotein (Wehner et al., 1997
). As an initial step to determine the roles of the p10 protein in the virus life-cycle, we analysed the interactions of the p10 protein with the BDV-specific phosphoprotein p24 and the nucleoprotein p38/p39 which are probably essential components of the virus replication machinery. Analysis of lysates from BDV-infected cells by anti-p10 immunoaffinity chromatography suggested that fractions of the p24 and p38 proteins form complexes with p10 that are stable at high salt concentrations. Moreover, the dissection of these proteinprotein interactions in glutathione agarose-based capture assays indicated that the BDV p10 protein interacts directly with the p24 phosphoprotein and indirectly with the viral nucleoprotein via bridging through p24. A detailed mutational analysis revealed that a short leucine-rich sequence in p10 at amino acids 815 is critical for binding to the viral phosphoprotein. Mutational inactivation of the p24 interacting domain resulted in failure of the p10 protein to associate with the BDV-specific intranuclear foci in infected cells. Due to the association with the predicted viral replicative phospho- and nucleoproteins, possible regulatory roles of the BDV p10 protein in viral RNA synthesis or transport are discussed.
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Methods |
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Yeast strains, Escherichia coli strains and plasmids.
E. coli strains used for cloning and expression were DH5 and BL26. Saccharomyces cerevisiae EGY48 (Mata trp1 ura3 his3 LEU2::pLEX-Aop6-LEU2), plasmids pSH18-34, pRFHM1, pJG4-5, pcDNA-p10 and pGEX-p10 have been described previously (Gyuris et al., 1993
; Wehner et al., 1997
). Construction of plasmids followed standard cloning procedures (Ausubel et al., 1992
). The plasmids pcDNA-p38 and pcDNA-p24 were prepared by subcloning BDV p38 cDNA (nucleotide positions 931166 according to Cubitt et al., 1994
) and p24 cDNA (nucleotides 12721877) between the BamHI and EcoRI/BamHI sites of pcDNA3 (Invitrogen), respectively. Plasmids pJG-p10, pGILDA-p10, pGILDA-p24 and pGILDA-p39 were generated by subcloning cDNAs encoding the p10 (nucleotides 12201486), p24 and p39 (nucleotides 541166) proteins of BDV strain He/80 between the EcoRI/XhoI sites of pJG4-5 and pGILDA (Clontech), respectively. Plasmids of the pGILDA series express fusion proteins of the bacterial LexA DNA-binding domain whereas pJG constructs encode fusions with the B42 acidic transactivation domain attached to an HA-tag sequence. Derivatives of pGILDA-p10 expressing C-terminally truncated p10 proteins were constructed by introduction of translational stop codons through site-directed mutagenesis using a Quikchange kit (Stratagene). Plasmids encoding N-terminally truncated p10 proteins were made by PCR amplification of corresponding p10 cDNAs and subcloning the resulting products between the EcoRI/XhoI sites of pGILDA. pGILDA-p10 plasmids encoding p10 proteins with di-alanine substitutions were generated by site-directed mutagenesis. The bacterial expression plasmids pGEX-p24 and pGEX-p10 120 were constructed by inserting the corresponding p24 and p10 cDNAs between the EcoRI/BamHI sites of pGEX-2T (Pharmacia) and the EcoRI/XhoI sites of pGEX-5X-1 (Pharmacia), respectively. p10 cDNA was amplified and subcloned between the EcoRI/SfuI sites of pcDNA3.1/Myc-His C (Invitrogen) to obtain a vector expressing a p10Myc fusion protein in mammalian cells. Within this construct, the internal ATG codon in the p10 gene that initiates translation of the overlapping p24 reading frame was inactivated by site-directed mutagenesis without alteration of the derived p10 amino acid sequence resulting in pcDNA-p10-Myc. A further modification that resulted in the replacement of leucine residues 7, 9 and 10 by alanines (pcDNA-p10-Myc-mut) was introduced by site-directed mutagenesis. The plasmids were confirmed by DNA sequencing using a cycle sequencing protocol followed by analysis through an automated DNA sequencer ABI 377 (Perkin Elmer).
Generation of anti-GSTp10 and anti-GSTp24 sera.
Glutathione S-transferase (GST)p10 and GSTp24 fusion proteins were affinity-purified on glutathione Sepharose resin (Pharmacia) from extracts of E. coli XL-1 Blue transformed with pGEX-p10 or pGEX-p24 as recommended by the manufacturer. Rabbit monospecific anti-GSTp24 serum was raised by several injections with purified GSTp24 fusion protein suspended in Freunds adjuvant. Similarly, anti-p10 serum was generated by several immunizations of a rabbit with purified GSTp10 fusion protein. The specificity of the sera was tested by immunoblot detection of the respective BDV antigens in homogenate of BDV-infected rat brain.
Anti-p10 immunoaffinity chromatography.
Anti-p10 immunoaffinity resin was prepared by covalent immobilization of immunoglobulins from rabbit anti-GSTp10 serum (Wehner et al., 1997 ) on pre-activated Sepharose CL-6B matrix (Pharmacia) according to the protocol supplied by the manufacturer. The serum did not cross-react with any other BDV-specific proteins to detectable levels. Monolayers of BDV-oligo cells were collected in PBS and lysed by sonication. Extracts were clarified by centrifugation at 20000 g for 15 min and cycled several times over the anti-p10 affinity column. Subsequently, the column was washed with PBS and eluted with PBS containing 1 M NaCl, 3 M NaCl or 1 M NaClO4. Eluate fractions of 2 ml were collected and assayed by anti-p10, anti-p24 and anti-p38/p39 immunoblotting as described previously (Wehner et al., 1997
).
Yeast two-hybrid analysis of proteinprotein interactions by a
-galactosidase plate assay.
To assay for interactions between BDV p10, p24 and p39 proteins, EGY48 yeast cells harbouring the LexA-dependent lacZ reporter plasmid pSH18-34 were transformed pairwise with plasmids of the pGILDA and pJG series using the lithium acetate method (Ausubel et al., 1992 ). Yeast colonies were streaked onto plates containing X-Gal and incubated at 30 °C as described previously (Ausubel et al., 1992
). The interactions between the vector-encoded proteins were assessed by blue colour development after 24 h. The expression of BDV fusion proteins was verified by immunoblot analysis of yeast cell extracts using MAbs recognizing the LexA DNA-binding domain and the vector-encoded HA-tag sequence fused to the activation domain, respectively.
Co-precipitation of BDV p24 and p38 proteins with GSTp10 by glutathione Sepharose.
The BDV p10 protein was expressed from pGEX-p10 as a GST fusion protein in E. coli BL26. Synthesis of GSTp10 was induced by addition of 1 mM IPTG. Bacterial cell lysate containing the GSTp10 fusion protein was adsorbed to glutathione Sepharose according to the protocol supplied by the manufacturer and contaminating proteins were removed by three washes with PBS. The BDV p24 and p38 proteins were synthesized and labelled with [35S]methionine in coupled 50 µl transcription/translation reactions (TNT; Promega) programmed with pcDNA-p24 and/or pcDNA-p38. The translation reactions were mixed with 10 µl coated glutathione Sepharose beads in 750 µl NET-N buffer (10 mM TrisHCl, pH 8·0, 1 mM EDTA, 150 mM NaCl, 0·05% Nonidet P-40) for 2 h at 4 °C. The beads were washed three times with PBS/0·01% Nonidet P-40 and the precipitated proteins were separated by SDS gel electrophoresis and visualized by autoradiography.
Indirect immunofluorescence microscopy.
BDV-Oligo cells were transfected with the plasmids pcDNA-p10-Myc wild-type or pcDNA-p10-Myc-mut using the DOTAP reagent (Boehringer Mannheim) and seeded on glass cover slips in DMEM containing 10% foetal calf serum. After 36 h, cells were processed for immunofluorescence analysis by fixation in 2·5% methanol-free formaldehyde (Polysciences) and permeabilization of cells in 0·1% Triton X-100 was done as described previously (Wolff et al., 1998 ). Cells were stained with primary antibodies (rabbit anti-GSTp24 serum, 1:400; Myc-specific MAb 9E10, 1:50) diluted in PBS/3% BSA. The cells were washed and incubated with FITC-conjugated goat anti-mouse immunoglobulin G (IgG) and Texas red-conjugated goat anti-rabbit IgG. Subsequently, the cover slips were washed and mounted in MOWIOL 4-88 (Calbiochem). For immunofluorescence analysis, cells were viewed on a Zeiss Axiophot fluorescence microscope using a 63x objective and photographs were captured by a SPOT video camera (INTAS).
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Results |
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Discussion |
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We have previously demonstrated that BDV expresses the p10 protein as a sixth viral gene product (Wehner et al., 1997 ). During the initial characterization, the p10 protein had been detected co-localized with the viral nucleoprotein in the nucleus of BDV-infected cells, suggesting an association with the virus replication complexes. Here, we have demonstrated that the p10 protein tightly associates with the viral phospho-and nucleoproteins in complexes that are stable under high salt conditions. Moreover, our two-hybrid and co-precipitation experiments strongly indicated that the p10 protein interacts directly with the viral phosphoprotein, as has been observed previously (Schwemmle et al., 1998
). We did not detect significant interaction of the p38/p39 nucleoprotein with the p10 protein in the absence of p24, in contrast to the findings of Malik et al. (1999)
. However, the viral nucleoprotein was efficiently co-precipitated with p10 in the presence of p24. Since it is known that p24 binds to p38/p39 (Berg et al., 1998
; Hsu et al., 1994
) and the interactive domains for the p10 and p38/p39 proteins have been located in different regions of the phosphoprotein (Schwemmle et al., 1998
), it is likely that p24 bridges an indirect association between p10 and the nucleoprotein. Alternatively, the binding of p24 may induce a conformational change in the p10 protein allowing association with the viral nucleoprotein to occur.
We have explored the p10p24 interaction through extensive mutational analysis in genetic and biochemical assay systems. As a result, we identified p10 amino acid positions 815 to be critical for efficient p24 interaction. The failure of a mutant p10 protein, inactive in p24 binding, to associate with the BDV-specific intranuclear foci suggested that it is mainly the interaction with the viral phosphoprotein which mediates integration of p10 into viral nucleocapsids. Interestingly, this p10 region is rich in leucines and resembles NES that mediate interactions with the cellular export machinery (reviewed by Mattaj & Englmeier, 1998 ). The BDV genomic RNA is replicated in the nucleus of infected cells. Thus, there is a need for bornaviruses to regulate nuclear import and export of the viral genome. Other viruses with a nuclear replication strategy like HIV or influenza viruses express the small NES-containing rev and NEP/NS2 proteins, respectively, that associate with the viral genomes and mediate their nuclear export through interactions with cellular export receptor proteins (reviewed by Whittaker & Helenius, 1998
). Based on the presence of a leucine-rich sequence at p10 positions 412, we and others have previously speculated that p10 may have a homologous function in nuclear export. Surprisingly, our mutational analysis suggested that the same region within the p10 protein engages in robust binding to the viral phosphoprotein. As a result, we expect p24 binding to prevent further interactions of p10 with third factors like export receptors. Preliminary quantification analyses suggested that there are considerably higher levels of p24 protein in persistently infected cells compared to p10 (Z. Mohammadi-Motahhari and T. Wolff, unpublished results). Therefore, disassembly of the p10p24 complex is probably required to make the N-terminal region of the p10 protein available for interactions with other factors. Clearly, further analysis will be needed in the future to assess potential functions of the p10 N-terminal domain in nuclear export.
We have previously reported that BDV expresses the p10 protein from a reading frame that initiates at an upstream start codon and overlaps with the coding region of the viral p24 phosphoprotein (Wehner et al., 1997 ). Thus, in terms of expression strategy, the BDV p10 protein shares common properties with the C proteins encoded by several paramyxo-and rhabdoviruses, although there is no noticeable sequence homology (Bellini et al., 1985
; Giorgi et al., 1983
; Spiropoulou & Nichol, 1993
). For the C proteins of Sendai virus and VSV, modulatory effects on viral RNA synthesis have been demonstrated in vitro and in vivo (Cadd et al., 1996
; Curran et al., 1992
; Horikami et al., 1997
; Latorre et al., 1998
; Peluso et al., 1996
; Tapparel et al., 1997
). The Sendai virus C protein may exert this regulatory function through direct binding to the L polymerase subunit (Horikami et al., 1997
). Once suitable assay systems become available for BDV, it will be interesting to address the question of whether the p10 protein, which associates with the predicted RNA polymerase co-factor p24, also has a regulatory function in viral RNA synthesis. We expect that the interaction analysis of the p10 protein will help us in exploring the architecture of the BDV replication complex in the future.
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Acknowledgments |
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References |
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Received 8 October 1999;
accepted 8 December 1999.