Neither phosphorylation nor the amino-terminal part of rabies virus phosphoprotein is required for its oligomerization

Benoit Gigant1, Frédéric Iseni2, Yves Gaudin3, Marcel Knossow1 and Danielle Blondel3

Laboratoire d’Enzymologie et Biochimie Structurales (LEBS), CNRS, 91198 Gif sur Yvette, France1
EMBL Grenoble Outstation c/o ILL PB156, 38042 Grenoble cedex, France2
Laboratoire de génétique des virus, CNRS, 91198 Gif sur Yvette, France3

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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Rabies virus (PV strain) phosphoprotein (P) was expressed in bacteria. This recombinant protein binds specifically to the nucleoprotein–RNA complex purified from infected cells. Chemical cross-linking and gel-filtration studies indicated that the P protein forms oligomers. Analytical centrifugation data demonstrated the co-existence of monomeric and oligomeric forms of rabies virus P protein and suggested that there is an equilibrium between these species. As P expressed in bacteria is not phosphorylated, this result indicates that P phosphorylation is not required for its oligomerization. Although an alignment of several rhabdovirus P sequences revealed that the amino-terminal domain of P has a conserved predicted propensity to form helical coiled coils, an amino-terminally truncated form of P protein, lacking the first 52 residues, was also shown to be oligomeric. Therefore, the amino-terminal domain of rabies virus P is not necessary for its oligomerization.


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Rhabdoviruses contain a single-stranded, negative-sense RNA genome (11–15 kb) that is tightly encapsidated by the viral nucleoprotein (N) to form a ribonucleoprotein (RNP), which serves as a template for transcription and replication. During transcription, a positive-stranded leader RNA and five capped and polyadenylated mRNAs are synthesized (Banerjee, 1987 ). The replication process yields nucleocapsids containing full-length antigenome-sense RNA, which in turn serve as templates for the synthesis of genome-sense RNA. The active virus-encoded RNA polymerase complex is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (Emerson & Wagner, 1972 ). The L protein is a multifunctional enzyme and is the RNA-dependent RNA polymerase. This polymerase complex carries out all of the enzymatic steps of transcription, including initiation and elongation of transcripts, as well as co-transcriptional modifications of RNAs such as capping and polyadenylation (Banerjee, 1987 ).

Studies with vesicular stomatitis virus (VSV) have shown that the P protein is a non-catalytic cofactor and a regulatory protein: it associates with the L protein in the polymerase complex and interacts with both soluble and genome-associated N protein (Emerson & Schubert, 1987 ; Masters & Banerjee, 1988 ; Takacs & Banerjee, 1995 ). VSV P protein has different phosphorylation states that are believed to bind to the RNP with different affinities and to have different transcription activities (Barik & Banerjee, 1992a , b ; Gao & Lenard, 1995 ). Furthermore, the VSV P protein has been shown to form oligomers and oligomerization seems to be necessary for binding both to the L protein and to the template (Gao et al., 1996 ). The oligomerization is dependent on the phosphorylation of two residues near the amino terminus (Ser60 and Thr62) (Gao et al., 1996 ). Substitution of these amino acids by aspartic acid residues renders the protein oligomeric and fully active without phosphorylation (Gao et al., 1996 ).

Rabies virus and VSV are structurally similar. Thus, by analogy, their RNA polymerase complexes may have similar properties. Studies in vitro and in vivo have shown that rabies virus P protein forms specific complexes with the N and L proteins (Chenik et al., 1994 , 1998 ; Fu et al., 1994 ). We have demonstrated previously the existence of two N protein-binding sites on the P protein: one is located between amino acids 69 and 177 and another is positioned in the carboxy-terminal region comprising amino acids 268–297 (Chenik et al., 1994 ). We have shown that the major L-binding site resides within the first 19 residues of P (Chenik et al., 1998 ). It has been shown recently that rabies virus P protein is phosphorylated by two kinases, a unique cellular protein kinase (RVPK) and specific isomers of protein kinase C (Gupta et al., 2000 ). Both kinases phosphorylate at specific sites on the P protein, resulting in the formation of distinct phosphorylated forms of P protein having different mobilities in SDS–PAGE. Four additional, smaller amino-terminally truncated products (PA2, PA3, PA4, PA5) translated from the P mRNA have been found in purified virus, in infected cells and in 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.

The absence of an in vitro transcription system for rabies virus has precluded the characterization of the role of the P protein in virus transcription and replication. As a first step toward this characterization, wild-type P protein was expressed in bacteria. For this purpose, the P gene was placed downstream of the T7 promoter between the NdeI and XhoI cloning sites in the E. coli expression vector pET-22b (+) (Novagen). The NdeI site (CATATG) overlaps the initiation methionine codon ATG of the P gene. At the carboxy terminus, the expressed P protein contains two additional amino acids (Leu–Glu) followed by six histidine residues to facilitate its purification. When bacteria containing the recombinant plasmid (pET-22-P) were induced with IPTG, a polypeptide accumulated that migrated in SDS–PAGE with an apparent molecular mass of 38–40 kDa, the same as that of rabies virus P protein (not shown). This protein was purified by nickel-affinity chromatography followed by chromatography on a DEAE-Tris-Acryl column. The recombinant protein was also recognized by a previously available polyclonal anti-P antibody (not shown), and is thus further designated P-his.

We have shown previously that the rabies virus P protein interacts with the N protein (Chenik et al., 1994 ). Here, we studied the ability of P-his to bind to the template N–RNA isolated from infected cells. The incubation of P-his with the nucleocapsid was followed by centrifugation through 20% sucrose and the analysis of the supernatant and the pelleted material by SDS–PAGE. Protein P-his, which was present mostly in the supernatant in the absence of N–RNA, was brought down in the pellet with the template (Fig. 1a). This interaction was specific, since BSA did not pellet with the N–RNA template (not shown). These results indicate that P-his binds specifically to N–RNA templates.



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Fig. 1. (a)–(b) N-binding activity of the P-his (a) and PA3-his (b) proteins. P-his and PA3-his were purified as follows. Cells were harvested by centrifugation and resuspended in binding buffer containing 5 mM imidazole, 500 mM NaCl and 20 mM Tris–HCl supplemented with antiprotease cocktail (2 µg/ml leupeptin, 2 µg/ml antipain, 2 µg/ml pepstatin, 2 µg/ml chymostatin, 16 µg/ml aprotinin). The cell suspension was sonicated and the lysate was centrifuged at 15000 g for 20 min. The supernatant containing the bacterial P or PA3 proteins was loaded on a column of His-bind resin charged with a solution of NiSO4 as described by the manufacturer (Novagen). The column was washed first with a buffer containing 60 mM imidazole, 250 mM NaCl and 10 mM Tris–HCl, pH 8, and then with 30 mM imidazole, 20 mM NaCl, 10 mM Tris–HCl, pH 8. The proteins were then eluted with 500 mM imidazole, 50 mM NaCl and 20 mM Tris–HCl, pH 8. After tenfold dilution in 20 mM Tris–HCl, pH 8, and 1 mM EDTA, the P protein was loaded on a DEAE Tris-Acryl column. The column was washed with 10 mM NaCl, 200 mM Tris–HCl, pH 8, and 10 mM EDTA. P-his and PA3-his elutions were then performed with 500 mM NaCl, 20 mM Tris–HCl, pH 7·5, 1 mM EDTA. N–RNA templates isolated from infected BSR cells as described by Iseni et al. (1998) were incubated with P-his (a) or PA3-his (b) in 150 mM NaCl, 20 mM Tris–HCl, pH 7·4, for 1 h at 4 °C. The mixture was then sedimented for 1 h at 40000 r.p.m. in a Beckman SW55 rotor with adaptors for small volumes through 0·5 ml 20% sucrose in 150 mM NaCl, 20 mM Tris–HCl, pH 7·4. Proteins present in the supernatant (S) and the pellet (P) were analysed by SDS–PAGE and stained with Coomassie blue. (c)–(d) Oligomeric status of P-his and PA3-his by cross-linking analysis. About 10 µg of P-his (c) and PA3-his (d) was incubated with the cross-linker EGS as shown. Reactions were stopped by the addition of glycine at 200 mM for 30 min at room temperature. Proteins were then analysed by SDS–PAGE (12%) and visualized by staining with Coomassie blue. Protein molecular mass standards are indicated. Species II and III are shown. The star indicates the higher oligomeric species. Lane V: purified rabies virus (PV strain).

 
In order to determine the oligomeric status of P-his, the protein was incubated with increasing concentrations of the cross-linking reagent ethylene glycol bis(succinimidyl succinate) (EGS) at final concentrations of 0·1, 0·5, 1 and 10 mM for 30 min at room temperature. The reactions were analysed by electrophoresis on a denaturing gel (Fig. 1c). P-his protein can be cross-linked into slower-migrating forms (indicated by II and III in Fig. 1c). A larger multimer (indicated by a star) is also observed. Based on their apparent molecular mass, species II (80 kDa) and III (120 kDa) have mobilities consistent with those expected for dimers and trimers, respectively. The larger multimer has a molecular mass slightly above 212 kDa and corresponds to a higher oligomeric form. The absence of intermediate forms between species III and the higher oligomeric form suggests that this higher oligomeric form is a dimer of species III. These results indicate that P-his is able to associate into oligomers.

The Stokes’ radius of purified P-his was determined by size-exclusion chromatography. P-his (loaded on a column at 1·5 mg/ml) was eluted as a single peak with a Stokes’ radius of 42·5±1  (Fig. 2a), much higher than the expected radius of a globular 33 kDa protein (about 20 ). Analysis of the sedimentation profile shows that three species constitute the major part of the P-his preparation (Fig. 2b). The major species had apparent sedimentation coefficients of 3·3±0·3 S (about 30% of the total protein) and 8·5±0·5 S (about 60% of the total protein); the sedimentation coefficient of the third species, the heaviest, could not be evaluated reliably because this species constituted less than 10% of the total protein. This result is consistent with P-his being an oligomer, with several co-existing species.



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Fig. 2. (a) Determination of the Stokes’ radii of P-his and PA3-his proteins by size-exclusion chromatography. Gel filtration was performed on a Superose 12 HR 10/30 column. Samples (100 µl) were run at 0·4 ml/min on the column in a buffer containing 10 mM Tris–HCl, pH 8, 150 mM NaCl, 1 mM EDTA. Standard proteins used to calibrate the column ({bullet}) were ribonuclease A (R, Rs=16·4 ), chymotrypsinogen A (C, Rs=20·9 ), ovalbumin (O, Rs=30·5 ), BSA (B, Rs=35·5 ), aldolase (A, Rs=48·1 ) and ferritin (F, Rs=61 ). The void volume, V0, was determined by measuring the elution volume with blue dextran. The Stokes’ radius was determined graphically on a plot of [-log(Kav)]0·5, where Kav is the molecular sieve coefficient [(Velution-V0)/(Vgel bed-V0)], versus Stokes’ radius, according to Siegel & Monty (1966) . (b) Analysis of P-his by sedimentation velocity. Sedimentation velocity experiments were performed at 20 °C with a Beckman Optima XL-A analytical centrifuge equipped with a Ti 60 titanium four-hole rotor with two-channel 12 mm path-length centre-pieces. Sample volumes of 400 µl were centrifuged at 60000 r.p.m. and radial scans of absorbance were taken at 6 min intervals. Data analysis was performed with the program SVEDBERG (Philo, 1994 ). The partial volume at 20 °C, 0·717 cm3/g, was calculated from the amino acid composition; the viscosity was taken as 1·002 centipoise and the solvent density as 1·005 g/cm3. P (0·4 mg/ml) was sedimented and data were recorded at 41, 51, 61, 71 and 81 min (from left to right). The fitted data curves are represented by solid lines. The inset represents the apparent distribution of the sedimentation coefficient obtained from the time derivative of the concentration profile (Stafford, 1992 ).

 
Thus, although this recombinant P protein is not phosphorylated, it is able to assemble into oligomers. This indicates that rabies virus P protein behaves like Sendai virus P protein, phosphorylation of which is not necessary for oligomerization (Curran et al., 1995 ; Tarbouriech et al., 2000 ), but differently from the wild-type phosphoprotein of vesiculoviruses, phosphorylation of which is required for the protein to be oligomeric and fully active (Gao & Lenard, 1995 ). The exact oligomeric status of the phosphoprotein of members of the Mononegavirales remains a matter of debate. The phosphoprotein of vesiculoviruses has been proposed successively to be a tetramer (Gao & Lenard, 1995 ), a trimer (Das et al., 1995 ; Gao et al., 1996 ) or a dimer (Chattopadhyay et al., 1997 ). The P protein of Sendai virus has been reported to be a trimer (Curran et al., 1995 ), but, more recently, Tarbouriech et al. (2000) have obtained convincing evidence that it is a tetramer. Our cross-linking data suggest that rabies virus P protein forms a trimer. However, they do not exclude completely the possibility that it forms a tetramer.

Our analytical centrifugation data demonstrate the co-existence of monomers and oligomers of rabies virus P and suggest that there may be an equilibrium between these species. Such an equilibrium has been demonstrated for VSV Indiana, for which exchange of monomers between assembled phosphorylated P protein trimers has been described (Gao et al., 1996 ). This equilibrium between a monomeric and an oligomeric form of the P protein explains some conflicting results on the transcriptional activity of non-phosphorylated VSV P. In many cases, transcriptional activity was not found at low concentrations of non-phosphorylated P but was restored at high concentrations of the protein (Spadafora et al., 1996 ). High P concentrations shift the equilibrium toward the oligomeric species, which is probably the transcriptionally active form of the protein. Thus, the role of phosphorylation would be to increase the association constant of the equilibrium so that sufficient amounts of the oligomeric form would be present under physiologically low concentrations of P. The experiments performed here to characterize the oligomeric status of rabies P were performed at high P concentrations (about 1 mg/ml, i.e. 30 µM), which certainly favoured oligomerization, and we cannot exclude the possibility that phosphorylation of rabies virus P protein also results in an increase in the association constant of the equilibrium between the monomeric and the oligomeric forms of rabies virus P protein.

An analysis by Curran et al. (1995) predicted that the most likely oligomerization domain in VSV P protein comprises residues 1–30. P protein sequences of rabies virus (PV strain) and four other rhabdoviruses (Chandipura, Piry, VSV Indiana and VSV New Jersey) were submitted to a coiled coil-prediction program (Fig. 3a). This analysis revealed that the amino-terminal region has a potential for coiled-coil formation (Fig. 3a, b). This propensity to form a coiled coil is conserved in a domain that bears little sequence conservation, although it is the most-conserved region of the protein among the Rhabdoviridae. As such structures are often involved in protein oligomerization, these putative coiled-coil domains are good candidates for the multimerization domain.



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Fig. 3. Computer predictions on rhabdoviruses P protein sequences. (a) Alignment of the amino-terminal regions of the P proteins of rabies virus (PV strain) and four vesiculoviruses: VSV New Jersey (NJ), VSV Indiana (IND), Piry and Chandipura. Positions a and d of the heptad repeats are indicated. (b) Probability of coiled-coil formation of rhabdovirus P proteins, scored with the program MacStripe 2.0b1 (written by Alex Knight; see http://www.york.ac.uk/depts/biol/units/coils/coilcoil.html). The elevated coiled-coil probability in the carboxy-terminal part of rabies virus P (not predicted for the other rhabdovirus P protein sequences) is an artefact of the detection program, which is biased towards hydrophilic, highly charged sequences. A careful look at the primary P sequence in this region does not reveal any heptad repeats.

 
In order to study the function of the predicted coiled-coil sequence, we expressed the PA3-his protein, which lacks the first 52 amino-terminal residues. This recombinant protein, containing two additional amino acids (Leu–Glu) followed by six histidine residues at its carboxy terminus, migrated with an apparent molecular mass of 29 kDa in SDS–PAGE and was also able to bind to N–RNA templates from infected cells (Fig. 1b). This is consistent with the localization of the N-binding sites described previously on P (Chenik et al., 1994 ). Cross-linking experiments with increasing concentrations of EGS indicated that, in addition to the monomeric form, two slower-migrating forms (II and III) were obtained with apparent molecular masses of 44 and 66 kDa, respectively (Fig. 1d).

The Stokes’ radius of purified PA3-his was also determined by size-exclusion chromatography. The protein eluted as a single peak with a Stokes’ radius of 40±2  (Fig. 2). Again, this value is much greater than the expected Stokes’ radius of a globular protein of 29 kDa. Together with the cross-linking experiments, these results are consistent with PA3 being an oligomer. Thus, the amino-terminal region is not essential for rabies virus P oligomerization, even though it has a predicted high propensity for coiled-coil formation.

In summary, we have shown that neither phosphorylation nor the amino-terminal part of P is required for oligomerization. Furthermore, our data indicate that an equilibrium exists between monomeric and oligomeric forms of the P protein. It is noteworthy that the retention of a significant amount of P protein in a monomeric form in the infected cell is a conserved feature, at least among members of the Rhabdoviridae. This suggests that the monomer of P protein has a role during the virus cycle. Clearly, the P protein is multifunctional. It is required for the polymerase activity of the virus but it also interacts with the N protein, maintaining the N protein in a soluble form competent to support efficient RNA encapsidation during replication. It is thus possible that this or another undefined role is played by the monomeric form of P.


   Acknowledgments
 
We thank Gérard Batelier for performing analytical centrifugation experiments. We are grateful to Rob Ruigrok for helpful discussions and careful reading of the manuscript and also for sharing results before publication, Anne Flamand for constant support and Nicolas Poisson for his help in drawing of the figures. This work was supported by CNRS (UPR 9053 and UPR 9063).


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Received 7 December 1999; accepted 24 March 2000.