Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, France1
Laboratoire des Lyssavirus, Institut Pasteur 25 rue du Dr Roux, 75724 Paris Cedex 15, France2
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3305, USA3
Author for correspondence: Danielle Blondel. Fax +33 1 69 82 43 08. e-mail Danielle.Blondel{at}gv.cnrs-gif.fr
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Abstract |
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Lyssavirus ribonucleoproteins (RNP) contain the genomic RNA tightly encapsidated by the viral nucleoprotein (N) and the RNA polymerase complex, consisting of the large protein (L) and its co-factor, the phosphoprotein (P) (Emerson & Wagner, 1972 ). Both L and P proteins are involved in transcription and replication. During transcription, a positive-stranded leader RNA and five mRNAs are synthesized. The replication process yields nucleocapsids containing full-length antisense genomic RNA, which in turn serves as a template for the synthesis of positive-sense genomic RNA.
RV P protein is a non-catalytic co-factor and a regulatory protein: it associates with the L protein in the polymerase complex and interacts with both soluble and genome-associated N proteins. We have demonstrated previously the existence of two N protein-binding sites on the P protein: one located between amino acids 69 and 139 and the other located in the carboxy-terminal region comprising amino acids 268 to 297 (Chenik et al., 1994 ). We have shown also that the major L-binding site resides within the first 19 residues of P (Chenik et al., 1998
). In addition, four other amino-terminally truncated products (PA2, PA3, PA4 and PA5) translated from P mRNA have been found in purified virus, infected cells and cells transfected with a plasmid encoding the complete P protein. Translation of these proteins is initiated from internal in-frame AUG initiation codons by a leaky scanning mechanism (Chenik et al., 1995
). Their potential role in the virus cycle remains to be determined.
We have identified recently the cytoplasmic dynein light chain LC8 as a strong interacting partner of the P protein of two lyssaviruses, RV and Mokola virus, in a yeast two-hybrid screen (Jacob et al., 2000 ; Raux et al., 2000
). The PLC8 interaction was confirmed both in cells transfected with a plasmid encoding the P protein and in infected cells by co-immunoprecipitation (Raux et al., 2000
). Co-localization of the two proteins was demonstrated also by confocal microscopy (Jacob et al., 2000
). Dynein is a microtubule-associated motor protein complex involved in minus end-directed movement of organelles along microtubules (Bowman et al., 1999; Pazour et al., 1998). The PLC8 interaction could explain the propagation of the virus from the site of entry, such as a bite on the skin, to the CNS, i.e. via the long nerve axons. The LC8 protein, which forms dimers (Benashski et al., 1997
; Liang et al., 1999
), is suggested also to be an inhibitor of neuronal nitric oxide synthase (nNOS) (Jaffrey & Snyder, 1996
) and NO changes in the CNS have been proposed to explain some of the neuropathogenic events occurring during RV infection (Akaike et al., 1995
). LC8 homologues from evolutionarily distant species, such as human, rat, fly, nematode, fruit and green alga, display a remarkable degree of sequence identity and are constitutively produced in various cell types (King et al., 1996).
In order to define precisely the LC8-binding domain on P, we first constructed a mutant lacking residues 139172 (PN139172), residues that were suspected to contain the LC8-binding site (Raux et al., 2000
; Jacob et al., 2000
). This mutant was tested for its ability to interact with LC8. Proteins from transfected cells were immunoprecipitated from cell extracts using a polyclonal anti-P antibody (Raux et al., 1997
). The proteins present in the immune complexes were then detected on a Western blot using a rabbit polyclonal anti-LC8 antibody (R4058) (King & Patel-King, 1995
). As expected, protein P interacted with the endogenous LC8, whereas P
N139172 did not (Fig. 1A
, lanes 1 and 2). However, P
N139172 was expressed efficiently (Fig. 1A
, lanes 4 and 5) and was able to bind to N protein in cells co-transfected with both plasmids, as shown after immunoprecipitation with the anti-P antibody (Fig. 1A
, lanes 6 and 7), suggesting that P
N139172 was correctly folded.
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Analysis of the sequences of the lyssavirus P proteins (RV strains CVS and PV or Mokola virus) has shown that a region from amino acids 139 to 151 is similar in sequence to other LC8-binding partners (Jacob et al., 2000 ). Thus, we constructed the deletion mutant P
N139151 in pCDM8 and pLex in order to test its ability to bind to LC8 by co-immunoprecipitation and with the two-hybrid system, as described above. The results obtained with both methods demonstrated that P
N139151 did not bind to LC8 (Fig. 1B
, lane 2 and Fig. 1C
), but interacted efficiently with the N protein (data not shown).
The structure of the LC8 protein bound to a 13 residue peptide from nNOS has been solved by X-ray diffraction (Liang et al., 1999 ). In the crystal structures, the nNOS peptide lies in a deep groove formed between the monomers of the LC8 dimer. Eleven residues of the peptide (227EMLDTGIQVDR237) participate as a
-strand structure in the formation of a central
-sheet comprising six anti-parallel
-strands. Four strands derive from one monomer, the fifth strand from the other monomer and the sixth from the peptide. Among these residues, D230 and Q234 make hydrogen bonds via their side chains with the LC8 dimer. These residues correspond to those present at positions 143 and 147 of the P sequence, which may then be important for the binding of P to the LC8 dimer.
We then carried out site-directed mutagenesis of residues D143 and Q147, altering either one or both residues. We expressed these P gene mutants in BSR cells and yeast and tested their interaction with LC8 by using the methods described above. As shown in Fig. 1(B), the substitution of the two residues D143 and Q147 with an A residue abolished binding to LC8 (Fig. 1B
, lane 3). This was confirmed by the quantitative
-galactosidase assay in the yeast two-hybrid system (Fig. 1C
). One substitution (Q147A) resulted in a substantial loss of interaction, as shown by immunoprecipitation (Fig. 1B
, lane 5), and this interaction was too weak to be detected by the quantitative
-galactosidase assay (Fig. 1C
). In contrast, protein P(D143A) bound to LC8, but less efficiently than the wild-type P protein (Fig. 1B
, lane 4). This was confirmed by the
-galactosidase assay (Fig. 1C
). These results suggest strongly that both residues, D143 and Q147, are critical for the binding of P to LC8, although the D to A substitution reduced only weakly the binding of P to LC8. This does not exclude the possibility that neighbouring amino acids could also be involved in stabilizing the interaction.
These results, taken together with the sequence alignment indicate that P and nNOS bind LC8 in a similar manner. Thus, the complex between the peptide residues S140-G150 of phosphoprotein P and LC8 was modelled using the structure of the complex between the 13 residue peptide of nNOS and LC8 (protein database code 1CMI) (Liang et al., 1999 ) as the starting point. The side chains of the amino acids from the sequence EMLDTGIQVDR of the peptide nNOS were substituted with the side chains of the sequence SSEDKSTQTTG of P(S140G150). The conserved amino acids D143 and Q147 were used to anchor the peptide and the conformations of their side chains were kept unmodified from those of the equivalent nNOS residues. The peptide P(S140G150) binds mainly to one monomer of the LC8 protein through the hydrogen-bonding scheme of the
-sheet and also through side chain interactions (Fig. 2A
). Despite the hydrophobic groove formed by the LC8 dimer and the polar properties of amino acids of the peptide P(S140G150) between D143 and Q147, no steric hindrance between the peptide and the LC8 dimer was observed (Fig. 2B
).
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In summary, we have defined precisely the LC8-binding domain on P between the amino acids at positions 139151 and demonstrated that two residues, D143 and Q147, are essential for this interaction. Other ligands of LC8 have been described recently and the LC8-binding sites identified (Puthalakath et al., 1999 ; Lo et al., 2001
). These sequences are similar to the one that we have defined on the RV P protein (Table 1
). Recently, a nuclear magnetic resonance structure of LC8 bound to the peptide from Bim, a Bcl-2 family member, was determined (Fan et al., 2001
) and binding of the sequence motif DKSTQ to LC8 is in accordance with our model proposed in Fig. 2
. Thus, it appears that the P protein binds to LC8 in a manner similar to many natural cellular partners of the protein.
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Acknowledgments |
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References |
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Benashski, S. E., Harrison, R. S., Patel-King, R. S. & King, S. M. (1997). Dimerization of the highly conserved light chain shared by dynein and myosin V. Journal of Biological Chemistry 272, 20929-20935.
Bourhy, H., Kissi, B. & Tordo, N. (1993). Molecular diversity of the Lyssavirus genus. Virology 194, 70-81.[Medline]
Chenik, M., Chebli, K., Gaudin, Y. & Blondel, D. (1994). In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. Journal of General Virology 75, 2889-2896.[Abstract]
Chenik, M., Chebli, K. & Blondel, D. (1995). Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. Journal of Virology 69, 707-712.[Abstract]
Chenik, M., Schnell, M., Conzelmann, K. K. & Blondel, D. (1998). Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. Journal of Virology 72, 1925-1930.
Emerson, S. U. & Wagner, R. R. (1972). Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. Journal of Virology 10, 1348-1356.
Fan, J.-S., Zhang, Q., Tochio, H., Li, M. & Zhang, M. (2001). Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain. Journal of Molecular Biology 306, 97-108.[Medline]
Guarente, L. (1993). Strategies for the identification of interacting proteins. Proceedings of the National Academy of Sciences, USA 90, 1639-1641.
Jacob, Y., Badrane, H., Ceccaldi, P. E. & Tordo, N. (2000). Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. Journal of Virology 74, 10217-10222.
Jaffrey, S. R. & Snyder, S. H. (1996). PIN: an associated protein inhibitor of neuronal nitric oxide synthase. Science 274, 774-777.
King, S. M. & Patel-King, R. S. (1995). Mr=8,000 and 11,000 outer arm dynein light chains from Clamydomonas flagella have cytoplasmic homologues. Journal of Biological Chemistry 270, 11445-11452.
Liang, J., Jaffrey, S. R., Guo, W., Snyder, S. H. & Clardy, J. (1999). Structure of the PIN/LC8 dimer with a bound peptide. Nature Structural Biology 6, 735-740.[Medline]
Lo, K. W. H., Naisbitt, S., Fan, J. S., Sheng, M. & Zhang, M. (2001). The 8-kDa dynein light chain binds to its targets via a conserved (K/R)XTQT motif. Journal of Biological Chemistry 276, 14059-14066.
Nejmeddine, M., Trugnan, G., Sapin, C., Kohli, E., Svensson, L., Lopez, S. & Cohen, J. (2000). Rotavirus spike protein VP4 is present at the plasma membrane and is associated with microtubules in infected cells. Journal of Virology 74, 3313-3320.
Nicholls, A., Bharadway, R. & Honig, B. (1993). GRASP: graphical representation and analysis of surface properties. Biophysical Journal 64, 166-167.
Puthalakath, H., Huang, D. C. S., OReilly, L. A., King, S. M. & Strasser, A. (1999). The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Molecular Cell 3, 287-296.[Medline]
Raux, H., Iseni, F., Lafay, F. & Blondel, D. (1997). Mapping of monoclonal antibody epitopes of the rabies virus P protein. Journal of General Virology 78, 119-124.[Abstract]
Raux, H., Flamand, A. & Blondel, D. (2000). Interaction of the rabies virus P protein with the LC8 dynein light chain. Journal of Virology 74, 10212-10216.
Suomalainen, M., Nakano, M. Y., Keller, S., Boucke, K., Stidwill, R. P. & Greber, U. F. (1999). Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. Journal of Cell Biology 144, 657-672.
Tordo, N., Badrane, H., Bourhy, H. & Sacramento, D. (1993). Molecular epidemiology of lyssaviruses: focus on the glycoprotein and pseudogenes. Onderstepoort Journal of Veterinary Research 60, 315-323.[Medline]
Wallace, A. C., Laskowski, R. A. & Thornton, J. M. (1995). LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Engineering 8, 127-134.[Abstract]
Ye, G. J., Vaughan, K. T. & Roizman, B. (2000). The herpes simplex virus 1 UL34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane. Journal of Virology 74, 1355-1363.
Received 7 June 2001;
accepted 30 July 2001.