Laboratoire dEnzymologie 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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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 268297 (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 SDSPAGE. 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 (LeuGlu) 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 SDSPAGE with an apparent molecular mass of 3840 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 NRNA 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 SDSPAGE. Protein P-his, which was present mostly in the supernatant in the absence of NRNA, was brought down in the pellet with the template (Fig. 1a
). This interaction was specific, since BSA did not pellet with the NRNA template (not shown). These results indicate that P-his binds specifically to NRNA templates.
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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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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 130. 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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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.
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References |
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Received 7 December 1999;
accepted 24 March 2000.