Phosphoprotein of the Rinderpest Virus Forms a Tetramer through a Coiled Coil Region Important for Biological Function
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m400673200
ISSN1083-351X
AutoresAbdur Rahaman, Naryanaswamy Srinivasan, N. Shamala, M.S. Shaila,
Tópico(s)Mosquito-borne diseases and control
ResumoPhosphoprotein (P) of negative sense RNA viruses functions as a transcriptional transactivator of the viral polymerase (L). We report here the characterization of oligomeric P protein of rinderpest virus (RPV) and provide a structural basis for its multimerization. By size exclusion chromatography and dynamic light scattering analyses we show that bacterially expressed P protein exists as an oligomer, thus excluding the role of phosphorylation in P protein oligomerization. Gel filtration analyses of various parts of the P protein, also expressed in Escherichia coli, revealed that the predicted coiled coil region in the C-terminal domain is responsible for P protein oligomerization. Dynamic light scattering analysis confirmed the oligomeric nature of the coiled coil region of P. Chemical cross-linking analysis suggested that the C-terminal coiled coil region exists as a tetramer. The tetramer is formed by coiled coil interaction as shown by circular dichroism spectral analysis. Based on sequence homology, we propose a three-dimensional structure of the multimerization domain of RPV P using the crystal structure for multimerization domain of sendai virus (SeV) P as a template. Four-stranded coiled coil structure of the model is stabilized by a series of interactions predominantly between short nonpolar side chains emerging from different strands. In an in vivo replication/transcription system using a synthetic minigenome of RPV, we show that multimerization is essential for P protein function(s), and the multimerization domain is highly conserved between two morbilliviruses namely RPV and peste de petits ruminants virus. These results are discussed in the context of biological functions of P protein among various negative-stranded RNA viruses. Phosphoprotein (P) of negative sense RNA viruses functions as a transcriptional transactivator of the viral polymerase (L). We report here the characterization of oligomeric P protein of rinderpest virus (RPV) and provide a structural basis for its multimerization. By size exclusion chromatography and dynamic light scattering analyses we show that bacterially expressed P protein exists as an oligomer, thus excluding the role of phosphorylation in P protein oligomerization. Gel filtration analyses of various parts of the P protein, also expressed in Escherichia coli, revealed that the predicted coiled coil region in the C-terminal domain is responsible for P protein oligomerization. Dynamic light scattering analysis confirmed the oligomeric nature of the coiled coil region of P. Chemical cross-linking analysis suggested that the C-terminal coiled coil region exists as a tetramer. The tetramer is formed by coiled coil interaction as shown by circular dichroism spectral analysis. Based on sequence homology, we propose a three-dimensional structure of the multimerization domain of RPV P using the crystal structure for multimerization domain of sendai virus (SeV) P as a template. Four-stranded coiled coil structure of the model is stabilized by a series of interactions predominantly between short nonpolar side chains emerging from different strands. In an in vivo replication/transcription system using a synthetic minigenome of RPV, we show that multimerization is essential for P protein function(s), and the multimerization domain is highly conserved between two morbilliviruses namely RPV and peste de petits ruminants virus. These results are discussed in the context of biological functions of P protein among various negative-stranded RNA viruses. Rinderpest virus (RPV), 1The abbreviations used are: RPV, rinderpest virus; P protein, phosphoprotein; PBS, phosphate-buffered saline; PMD, P multimerization domain; ELISA, enzyme-linked immunosorbent assay; NTA, nitrilotriacetic acid; PCT, P C-terminal region; PNT, P N-terminal region; CAT, chloramphenicol acetyltransferase; SEC, size exclusion chromatography; SeV, sendai virus; PPRV, peste de petits ruminants virus; DLS, dynamic light scattering. which causes rinderpest disease in large and small ruminants is an enveloped virus belonging to the morbillivirus genus of the family Paramyxoviridae. The negative sense, single-stranded RNA genome codes for six structural proteins: namely nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L). The viral N-RNA i.e. genomic RNA encapsidated with N protein is transcribed and replicated by the L (RNA-dependent RNA polymerase) and P complex (1Curran J. Kolakofsky D. Adv. Virus Res. 1999; 54: 403-422Crossref PubMed Google Scholar). The L protein is associated with N-RNA template through its interaction with P protein to form the transcribing ribonucleoprotein (RNP) complex. In addition to polymerization activity, L exhibits a number of other enzymatic activities including methyl transferase, 5′-cap synthesis of mRNA, and poly(A)+ polymerase (2Sedlmeier R. Neubert W.J. Adv. Virus Res. 1998; 50: 101-139Crossref PubMed Google Scholar). During transcription, the intergenic start/stop signals are recognized by polymerase complex resulting in the synthesis of monocistronic, capped, and polyadenylated mRNAs. Once the intracellular concentration of viral proteins reaches a threshold level, genome replication begins. The intracellular concentration of unassembled N protein (N0) is believed to regulate the switch from transcription to replication (3Lamb R.A. Kolakofsky D. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven Press, New York1996: 1177-1204Google Scholar). During replication, the same polymerase complex ignores stop signals and generates full-length unmodified encapsidated antigenomic RNA to serve as the template for the synthesis of progeny viral genomes. P proteins of negative-stranded RNA viruses play multiple roles during viral infection. They act as a transcriptional transactivator and recruit L protein onto viral N-RNA template (1Curran J. Kolakofsky D. Adv. Virus Res. 1999; 54: 403-422Crossref PubMed Google Scholar, 4Gao Y. Lenard J. J. Virol. 1995; 69: 7718-7723Crossref PubMed Google Scholar). P proteins also bind to the N-RNA template, independent of its role in the L-P polymerase complex, and activate transcription (5Curran J. Virology. 1996; 221: 130-140Crossref PubMed Scopus (70) Google Scholar). In addition to binding with the assembled nucleocapsid structure of the N-RNA template, P proteins interact with unassembled N proteins and prevent nonspecific aggregation of the latter by forming the N0-P complex, a precursor for encapsidating newly synthesized RNA during replication (6Curran J. Marq J.B. Kolakofsky D. J. Virol. 1995; 69: 849-855Crossref PubMed Google Scholar). P proteins of mononegalovirales undergo phosphorylation in one or more serine residues, which has been shown to be important for its function (7Pattnaik A.K. Hwang L. Li T. Englund N. Nathur M. Das T. Banerjee A.K. J. Virol. 1997; 71: 8167-8175Crossref PubMed Google Scholar, 8Dupuy L.C. Dobson S. Bitko V. Barik S. J. Virol. 1999; 73: 8384-8392Crossref PubMed Google Scholar). Although P proteins function as a homo-oligomer, their oligomerization status as well as the requirement of phosphorylation for oligomerization has been shown to vary among them (1Curran J. Kolakofsky D. Adv. 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Chem. 1995; 270: 24100-24107Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Chattopadhyay D. Raha T. Chattopadhyay D. Virology. 1997; 239: 11-19Crossref PubMed Scopus (24) Google Scholar). P proteins of all the paramyxoviruses harbor a coiled coil region at the C-terminal domain, and this region has been shown to be important for oligomerization in a number of viruses in the Paramyxoviridae family (13Tarbouriech N. Curran J. Ebel C. Ruigrok R.W. Burmeister W.P. Virology. 2000; 266: 99-109Crossref PubMed Scopus (82) Google Scholar, 18Shaji D. Shaila M.S. Virology. 1999; 258: 415-424Crossref PubMed Scopus (29) Google Scholar, 19Harty R.N. Palese P. J. Gen. Virol. 1995; 76: 2863-2867Crossref PubMed Scopus (82) Google Scholar, 20Chaoudhary S.K. Malur A.G. Huo Y. De B.P. Banerjee A.K. Virology. 2002; 302: 373-382Crossref PubMed Scopus (25) Google Scholar, 21Tarbouriech N. Curran J. Ruigrok R.W. Burmeister W.P. Nat. Struct. Biol. 2000; 7: 777-781Crossref PubMed Scopus (157) Google Scholar). The P protein has a modular structure, which comprises two major domains: the N-terminal domain is highly variable among various paramyxoviruses whereas the C-terminal domain, though exhibiting low sequence similarity, is conserved in terms of secondary structure (13Tarbouriech N. Curran J. Ebel C. Ruigrok R.W. Burmeister W.P. Virology. 2000; 266: 99-109Crossref PubMed Scopus (82) Google Scholar). The C terminus has been shown to have two subdomains in the sendai virus P protein: PMD, corresponding to the N-terminal region of the C-terminal domain that harbors the multimerization domain along with the L binding domain, and Px, corresponding to the rest of the C-terminal domain involved in nucleocapsid binding (13Tarbouriech N. Curran J. Ebel C. Ruigrok R.W. Burmeister W.P. Virology. 2000; 266: 99-109Crossref PubMed Scopus (82) Google Scholar). Earlier work on the RPV P protein has shown that while the first 59 amino acid residues at the N terminus along with the predicted coiled coil region in the C-terminal half are important for interaction with unassembled N protein, the last 17 amino acid residues along with the predicted coiled coil region are required for interaction with the nucleocapsid structure (18Shaji D. Shaila M.S. Virology. 1999; 258: 415-424Crossref PubMed Scopus (29) Google Scholar). This study also indicated the importance of the coiled coil region in P protein self-interaction. As a first step toward understanding the structure-function relationship of RPV P protein, we have looked at the oligomerization status of RPV P protein and examined the importance of coiled coil region of P protein in oligomerization as well as its function using different biochemical and biophysical approaches. Further, we propose a three-dimensional structure for the multimerization domain of RPV P based on its sequence similarity with that of SeV whose crystal structure has recently been solved (21Tarbouriech N. Curran J. Ruigrok R.W. Burmeister W.P. Nat. Struct. Biol. 2000; 7: 777-781Crossref PubMed Scopus (157) Google Scholar). The importance of phosphorylation and oligomerization of P protein in the transcription and replication processes of negative sense RNA viruses has also been examined. Materials—Escherichia coli DH5α strain was used for the maintenance of plasmids whereas the BL21 (DE3) strain was used for the expression of recombinant proteins (Invitrogen). p3e and p4a harboring the 1-291 (PNT) and 292-508 (PCT) amino acid regions of the P protein, respectively, were earlier cloned in the laboratory in pRSET vector whereas the full-length P (508 amino acids) gene isolated from a cDNA library of RPV (RBOK strain) was cloned in the expression vector pRSETB and designated pRP6 (22Kaushik R. Shaila M.S. J. Gen. Virol. 2004; 85: 687-691Crossref PubMed Scopus (27) Google Scholar). Plasmids pKSN-1 (RPV N gene in pBS), pPol10 (RPV L gene in pGEM), and pMDB8A (an RPV minigenome plasmid carrying the 3′-regulatory sequence; i.e. leader region, transcription/replication start regions, and 5′-trailer sequences flanking the CAT reporter gene open reading frame driven by the T7 promoter, T7 terminator, and δ ribozyme) were kindly provided by Dr. M. D. Baron, Institute of Animal Health, Pirbright, UK (23Baron M.D. Barrett T. J. Virol. 1997; 71: 1265-1271Crossref PubMed Google Scholar). A plasmid harboring the P gene of peste de petits ruminants virus (PPRV P in pGEM vector) was a gift from Dr. T. Barrett, Institute for Animal Health, Pirbright, UK. A549 cells derived from the Human Lung Carcinoma cell line were from ATCC. These cells were maintained on HAM12 containing 10% newborn calf serum (Invitrogen). VTF7-3 recombinant vaccinia virus expressing T7 polymerase in mammalian cells was a kind gift from Dr. Bernard Moss, National Institutes of Health. Cloning of the Coiled Coil Region (RPC), the Extreme C-terminal Region (Px) of RPV P, and the Multimerization Domain (PPMD) of PPRV P—The nucleotide sequence corresponding to RPC (amino acids 316-382), a part of the RPV PMD (amino acids 266-388) was released from pRP6 plasmid DNA by digestion with EcoRV and BamHI, and the end-filled insert was subcloned into NcoI- and XhoI-digested pET33b (+) vector after end-filling. The expressed protein from this clone gives nine additional amino acids, one (Met) at the N terminus and eight (Leu, Glu, and His6) at the C terminus. The Px (amino acids 377-508) was cloned by removing the NheI and SmaI fragment of pRP6 followed by religation of the backbone. The expressed protein results in 14 additional amino acids (MRGSH6GMAR) at its N terminus. The nucleotide sequence corresponding to the PPRV PMD region, PPMD (amino acids 264-387), was amplified using pTB-P DNA as template and appropriate primers (Forward, 5′-790CGA AAT GCG TCT GTG G805-3′, Reverse, 5′-TTA 1161CTC AGA TGT TGG GTC1147-3′). Nucleotide positions of the primers on the PPRV P gene are indicated within parentheses. The PCR product was cloned in the EcoRV site of pET20b (+) vector. The insert from the recombinant was released using NcoI and XhoI and subcloned into similar restriction sites of pET33b (+). The expressed protein codes for two additional amino acids (MD) at the N terminus. A stop codon was incorporated in the reverse primer to eliminate additional amino acids at the C terminus. Expression and Purification of Recombinant Proteins—E. coli BL21 (DE3) strain was transformed with plasmids carrying full-length as well as different parts of the RPV P. The transformant was grown in LB containing 100 μg/ml ampicillin (except for RPC) or 50 μg/ml kanamycin (for RPC) and induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside at an OD600 of 0.6 and grown for another 5 h. The cells were harvested and lysed by sonication in MCAC buffer (500 mm NaCl in 20 mm Tris-HCl, pH 8) and supplemented with 2 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture. The lysates were centrifuged, and supernatant was mixed with Ni-NTA agarose. The resin was washed with 100 bed volumes of MCAC buffer containing 50 mm imidazole except for RPC in which imidazole was not used. Proteins were eluted with 500 mm imidazole in MCAC supplemented with a protease inhibitor mixture. The RPC so obtained was dialyzed against 50 mm Tris-Cl, pH 8.0 and further purified by passing through a 5-ml QSepharose column using 0-300 mm NaCl in dialysis buffer as the gradient. Eluted samples of purified proteins were detected by Coomassie Blue staining of SDS-polyacrylamide gels. The protein concentration was measured by taking absorbance measurements at 280 nm, with the exception of RPC where the concentration was estimated by the Bradford assay. The identity of the proteins was confirmed by Western blot analysis using polyclonal antibodies raised in rabbit against bacterially expressed RPV P or PPRV P protein. Size Exclusion Chromatography (SEC)—Either the Sephadex G75 column (45 cm × 2.22 cm2, bed volume of 100 ml) or Sephacryl S300 column (60 cm × 2 cm2, 120-ml bed volume) was equilibrated with PBS or MCAC buffer, respectively, and calibrated using standard protein molecular mass markers. One milligram each of P, PNT, and PCT in MCAC or RPC and Px in 1 ml of PBS were separated on Sephacryl S300 or Sephadex G75, respectively, and the elution profiles were monitored by measuring the absorbance at 280 nm, except for RPC, which was monitored by protein estimation using the Bradford assay. The proteins were then identified using SDS-polyacrylamide gels and silver staining. Dynamic Light Scattering (DLS) Analysis—About 1 mg/ml of RPC (in 50 mm Tris-Cl, pH 8.0 and 50 mm NaCl) or 0.5 mg/ml of P (in 20 mm Tris-Cl, pH 8.0, and 500 mm NaCl) was subjected to DLS analysis using the DynaPro machine (Protein Solutions). About 50 observations were made to calculate the hydrodynamic radius (Rh) using DynaPro software. The viscosity used for Rh calculation was estimated from the refractive index of the buffer as measured by refractometer. Chemical Cross-linking—About 20 μg of purified RPC protein were cross-linked using glutaraldehyde (final concentration of 0.5 and 1 mm) for different time intervals from 30 min to4hat25 °C. The reaction was stopped by addition of 200 mm glycine, and the products were electrophoresed on a 15% SDS-polyacrylamide gel and detected by silver staining. Circular Dichroism (CD) Spectroscopy—Purified RPC at 0.1 mg/ml in PBS or in 50% trifluoroethanol in PBS was analyzed in a spectropolarimeter (JASCO J-715) at room temperature. The CD spectrum was measured in a cuvette of 2-mm path length, with a bandwidth of 0.5 nm and a scan speed of 50 nm/s. The buffer spectrum was subtracted from the protein spectrum. An average of four independent measurements were used to calculate molar residue ellipticity [θ]MRW using Equation 1,[θ]MRW=(θ⋅100⋅Mr)/(c⋅l⋅NA)(Eq. 1) where [θ] is the mean residue molar ellipticity in deg cm2 dmol-1, θ is experimental ellipticity in millidegree, Mr is the molecular weight of the protein, c is protein concentration in mg/ml; l is cuvette path length in centimeters, and NA is the number of residues of the protein. The percent helicity was estimated in Equation 2 (24Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3331) Google Scholar, 25Wu C.S.C. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar),% helicity=([θ]222−0[θ]222)/(100[θ]222−0[θ]222)×100(Eq. 2) where [θ]222 is the experimentally observed absolute mean residue ellipticity at 222 nm and values for 100[θ]222 and 0[θ]222, corresponding to 100 and 0% helix content at 222 nm, were estimated at 32,000 and 2,000 deg·cm2/dmol, respectively (25Wu C.S.C. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar, 26Chen Y.H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1971) Google Scholar). In Vivo Replication/Transcription Assay—To assess the significance of tetramerization on the biological function of the P protein, an in vivo replication-transcription assay using the minigenome construct pMDB8A was performed as described earlier (23Baron M.D. Barrett T. J. Virol. 1997; 71: 1265-1271Crossref PubMed Google Scholar). The transcript from the minigenome is antigenomic sense, which is replicated to genomic sense RNA by the virus proteins, L, N, and P; expressed by co-transfected plasmids in A549 cells infected with recombinant vaccinia virus expressing T7 RNA polymerase. The newly made genomic RNA was then transcribed into CAT mRNA, and the translated CAT protein was measured by ELISA (Roche Applied Science). A549 cells (1 × 106 cells/35-mm dish) were infected with recombinant vaccinia virus, VTF7-3 at a multiplicity of infection of 10 at 37 °C. At 1-h postinfection, the cells were washed with PBS and transfected using 5 μl of LipofectAMINE (2 mg/ml) in 1 ml of OPTI-MEM medium (Invitrogen) containing 1 μg each of pMDB8A, pKS-N, pRP6, and 100 ng of pGEM-L with or without pRPC/pPPMD. At 48-h post-transfection, the cells were harvested, and CAT activity was assayed by ELISA. Co-expression of Full-length RPV P with the PPRV P Multimerization Domain—The plasmid DNA of pRP6 and pPPMD clones were co-transformed into BL21 (DE3) strains of E. coli, and the recombinant cells harboring both plasmids were selected using two antibiotics, i.e. ampicillin (100 μg/ml) and kanamycin (50 μg/ml). Transformed cells were grown in Luria Broth supplemented with 100 μg/ml of ampicillin and 50 μg/ml of kanamycin to an OD600 of 0.6 at 37 °C. Expression and the purification of the protein by Ni-NTA agarose affinity chromatography were done as described above. The purity of both the purified proteins was tested by electrophoresis on a 15% SDS-polyacrylamide gel followed by Coomassie Blue staining and confirmed by Western blot analysis using the appropriate antibody. Prediction of Secondary Structures and Coiled Coil Regions—The sequence of the multimerization domain of RPV P-protein (PMD) was subjected to secondary structure prediction analysis using PHD as well as coiled coil region prediction (27Rost B. Sander C. J. Mol. Biol. 1993; 232: 584-599Crossref PubMed Scopus (2656) Google Scholar, 28Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar, 29Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3482) Google Scholar, 30Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (593) Google Scholar, 31Wolf P. Kim P.S. Berger B. Protein Sci. 1997; 6: 1179-1189Crossref PubMed Scopus (649) Google Scholar). These predictions were employed in order to get views about the potential of this region to adopt α-helical structure as well as to form coiled coils, independent of the fact that a distant homologue (sendai virus phosphoprotein) has the same structural features. Comparative Modeling of the Coiled Coil Region of the P Protein—The amino acid sequence of RPV PMD protein (amino acids 266-388) was aligned with that of the sendai virus phosphoprotein whose crystal structure shows a homotetrameric α-helical coiled coil structure (21Tarbouriech N. Curran J. Ruigrok R.W. Burmeister W.P. Nat. Struct. Biol. 2000; 7: 777-781Crossref PubMed Scopus (157) Google Scholar). The two proteins are distantly related, and the alignment is non-trivial. Hence the structural features (such as solvent accessibility and secondary structure) at every residue were evaluated, and relationships such as hydrogen-bonding patterns in the crystal structure were assessed. While aligning the sequences the probability of a residue in the RPV PMD protein adopting the structural environment of equivalent residues in the known structure was considered. The positive matches between predicted secondary structures in the P protein and the observed secondary structures in the crystal structure during alignment were also given importance. The suite of programs encoded in COMPOSER and incorporated in SYBYL (Tripos Inc., St. Louis) was used to generate a three-dimensional model of the P-protein (32Srinivasan N. Blundell T.L. Protein Eng. 1993; 6: 501-512Crossref PubMed Scopus (108) Google Scholar). The COMPOSER-generated model was energy-minimized in SYBYL using the AMBER force field (33Weiner S.I. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4895) Google Scholar). The energy-minimized model of a subunit of RPV PMD was superimposed with each one of the four subunits of SeV PMD, and the preliminary model for RPV PMD tetramer, so obtained, was subjected to further energy minimization to optimize interprotomer interactions. Recombinant Proteins—All recombinant proteins such as full-length P (amino acids 1-508), PNT (amino acids 1-291), PCT (amino acids 292-508), RPC (amino acids 316-382), and Px (amino acids 376-508) were expressed and purified to near homogeneity (Fig. 1). The authenticity of the purified proteins was confirmed by Western blot analysis using polyclonal antibody made against purified P protein expressed in E. coli (data not shown). As shown in Fig. 1, the full-length P and PNT migrate at positions corresponding to 80 and 52 kDa, respectively, which are much higher than their calculated masses (62 and 39 kDa, respectively). This anomalous mobility is attributed to the cluster of acidic residues at the N-terminal domain (18Shaji D. Shaila M.S. Virology. 1999; 258: 415-424Crossref PubMed Scopus (29) Google Scholar). Mass spectroscopic analysis of full-length P protein further confirmed its authenticity (data not shown). P Protein Exists as a Homo-oligomer in Solution—The oligomerization state of bacterially expressed P protein was studied by SEC. As shown in Fig. 2, the majority of the P protein elutes at a position that corresponds to a molecular mass of more than 300 kDa. This result indicates that P forms a higher order multimer because the monomeric molecular mass is 62 kDa. The hydrodynamic radius of P protein was measured by DLS. The Rh of 7 nm for the P protein confirms the formation of an oligomer in solution. The chemical cross-linking experiment also suggested that the P protein exists as a multimer (data not shown). Coiled Coil Region on the C-terminal Domain Is Responsible for Oligomerization of P Protein into a Tetramer—Recombinant proteins corresponding to the various parts of the P protein were subjected to SEC. Elution profiles are shown in Fig. 3. PNT (mass ∼39 kDa) elutes at around 100 kDa, indicating that it is either an oligomer or is a partially structured monomer. Earlier work had revealed that the C terminus is involved in P protein self-interaction (18Shaji D. Shaila M.S. Virology. 1999; 258: 415-424Crossref PubMed Scopus (29) Google Scholar). In the measles virus P protein, the equivalent domain (PNT) has been shown to be a partially structured monomer (34Karlin D. Longhi S. Receveur V. Canard B. Virology. 2002; 296: 251-262Crossref PubMed Scopus (90) Google Scholar). Further, PCT (mass ∼28 kDa) eluted from the gel filtration column at a position of molecular mass 150 kDa. Taken together, these results suggest that the oligomerization domain lies at the C terminus of P (PCT). The coiled coil region (RPC) and the rest of the C-terminal domain (Px) show molecular masses of 35 and 23 kDa, respectively, in SEC. This suggests that RPC (mass ∼8.5 kDa) is an oligomer, possibly a tetramer. The molecular size of Px (mass ∼16 kDa) is too small to be a dimer, and the increased molecular size of the monomer might result from its elongated shape or partially structured nature. The oligomeric state of RPC was further tested by DLS. The result showed an Rh of 2.8 nm (corresponding to ∼34 kDa) again confirming the oligomeric nature of RPC. To find out the exact stoichiometry of the RPC, chemical cross-linking of RPC was carried out. As shown in Fig. 4, in addition to monomers, cross-linked RPC was detected as dimers, trimers, and tetramers. With an increase in the duration of reaction and increase in cross-linker concentration, an increase in the number of tetramers was observed. Since cross-linked products higher than tetramer were not observed, we conclude that the most common form of RPC is a tetramer. The nature of interaction of such a tetramer was studied by CD spectral analysis in the presence and absence of trifluoroethanol (Fig. 5). These results indicated that RPC is rich in α-helical content (∼90%), and the ratio of ellipticities at 222/208 nm is greater than 1.0, indicative of the presence of interacting helices. Moreover, the ratio of ellipticities at 222/208 nm in 50% trifluoroethanol decreased to 0.918, a characteristic of non-interacting α-helices. Because trifluoroethanol has been shown to disrupt tertiary structure and quaternary structure and to promote secondary structure (35Lau S.Y. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 259: 13253-13261Abstract Full Text PDF PubMed Google Scholar), this result suggests that RPC forms a coiled coil structure. Taken together, these results lead us to conclude that RPV P protein forms a tetramer through coiled coil interaction present in RPC.Fig. 5Coiled coil interaction of RPC. CD spectra of 20 μm RPC in PBS (open circle) and 20 μm RPC in the presence of 50% trifluoroethanol in PBS (filled circle). The ratio of ellipticities 222/208 nm is greater than 1.0 indicating the presence of helix-helix interaction, i.e. coiled coil interaction. A ratio of less than 1.0 (0.918) in the presence of trifluoroethanol suggests non-interacting helices due to disruption of quaternary structure, i.e. coiled coil structure of RPC.View Large Image Figure ViewerDownload (PPT) P Protein Functions as a Multimer, and the Multimerization Domain Is Conserved between Two Morbilliviruses—The biological function of the multimerization domain of P protein was assessed employing an in vivo replication/transcription system for RPV. As shown in Fig. 6a, the CAT protein level is significantly reduced when RPC is coexpressed with wild-type P protein compared with the control where the full-length P plasmid alone was used. Earlier studies in our laboratory have revealed that the coiled coil region does not interact with N protein (18Shaji D. Shaila M.S. Virology. 1999; 258: 415-424Crossref PubMed Scopus (29) Google Scholar) or L protein (36Chattopadhyay A. Shaila M.S. Virus Genes. 2004; 28: 169-178Crossref PubMed Scopus (22) Google Scholar). These results clearly suggest that RPC forms a hetero-oligomer with wild-type P protein and
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