Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M400673200 on March 22, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23606-23614, May 28, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/22/23606    most recent
M400673200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rahaman, A.
Right arrow Articles by Subbarao Shaila, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rahaman, A.
Right arrow Articles by Subbarao Shaila, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Phosphoprotein of the Rinderpest Virus Forms a Tetramer through a Coiled Coil Region Important for Biological Function

A STRUCTURAL INSIGHT*

Abdur Rahaman{ddagger}§, Naryanaswamy Srinivasan¶||, Narayanaswamy Shamala**, and Melkote Subbarao Shaila{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Microbiology and Cell Biology, the Molecular Biophysics Unit, and the **Department of Physics, Indian Institute of Science, Bangalore 560012, India

Received for publication, January 21, 2004 , and in revised form, March 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rinderpest virus (RPV),1 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 (1). 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 (2). 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 (3). 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 (1, 4). P proteins also bind to the N-RNA template, independent of its role in the L-P polymerase complex, and activate transcription (5). 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 (6). P proteins of mononegalovirales undergo phosphorylation in one or more serine residues, which has been shown to be important for its function (7, 8). 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 (1, 9-17). 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 (13, 18-21). 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 (13). 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 (13). 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 (18). 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 (21). The importance of phosphorylation and oligomerization of P protein in the transcription and replication processes of negative sense RNA viruses has also been examined.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Escherichia coli DH5{alpha} 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 (22). 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 {delta} ribozyme) were kindly provided by Dr. M. D. Baron, Institute of Animal Health, Pirbright, UK (23). 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-{beta}-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 x 2.22 cm2, bed volume of 100 ml) or Sephacryl S300 column (60 cm x 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 [{theta}]MRW using Equation 1,

(Eq. 1)

where [{theta}] is the mean residue molar ellipticity in deg cm2 dmol-1, {theta} 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 (24, 25),

(Eq. 2)

where [{theta}]222 is the experimentally observed absolute mean residue ellipticity at 222 nm and values for 100[{theta}]222 and 0[{theta}]222, corresponding to 100 and 0% helix content at 222 nm, were estimated at 32,000 and 2,000 deg·cm2/dmol, respectively (25, 26).

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 (23). 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 x 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 (27-31). These predictions were employed in order to get views about the potential of this region to adopt {alpha}-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 {alpha}-helical coiled coil structure (21). 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 (32). The COMPOSER-generated model was energy-minimized in SYBYL using the AMBER force field (33). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (18). Mass spectroscopic analysis of full-length P protein further confirmed its authenticity (data not shown).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 1.
Expression and purification of recombinant deletion proteins of P. Polyacrylamide gels showing clarified cell lysate (lane 1), unbound fraction of the Ni-NTA column (lane 2), and purified sample (lane 3) of pRP6 (a), PNT (amino acids 1-291) (b), PCT (amino acids 292-508) (c), RPC (amino acids 316-382) (d), and Px (amino acids 377-508) (e). Molecular masses of the respective proteins were calculated using the standard low molecular mass markers obtained from Amersham Biosciences and are indicated by an arrow.

 
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).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 2.
Gel filtration analysis of pRP6. Nickel affinity-purified protein was fractionated through a Sephacryl S300 column (bed volume, 120 ml) and protein content in each fraction (1 ml each) was monitored by monitoring the absorbance at 280 nm. The gel filtration column was calibrated using the standard molecular mass markers for size exclusion column chromatography obtained from Amersham Biosciences, and the positions of different standard molecular mass markers are indicated on the top. The larger peak corresponds to >300 kDa, whereas the small peak corresponds to ~68 kDa.

 
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 (18). In the measles virus P protein, the equivalent domain (PNT) has been shown to be a partially structured monomer (34). 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).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3.
Elution profile of PCT (a), PNT (b), Px (c), and RPC (d) in gel filtration column. Purified proteins at 1 mg/ml were fractionated either on a Sephacryl S300 column (bed volume, 120 ml) or on a Sephadex G75 column (bed volume, 100 ml) as mentioned earlier. Protein content in each fraction (1 ml each) was monitored by A280 except RPC, which was monitored by estimating protein content of the fractions by Bradford assay. PCT, PNT, Px, and RPC eluted from the column at positions corresponding to 150, ~100, 23, and 35 kDa, respectively. The columns were calibrated as described in the text. The positions of different standard molecular mass markers are indicated on the top.

 
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 {alpha}-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 {alpha}-helices. Because trifluoroethanol has been shown to disrupt tertiary structure and quaternary structure and to promote secondary structure (35), 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.



View larger version (81K):
[in this window]
[in a new window]
  		FIG. 4.
Chemical cross-linking of RPC. Purified protein was dialyzed in PBS, and 10 µg of protein was incubated with either 0.5 mM glutaraldehyde (lanes 2-5) or 1 mM glutaraldehyde (lanes 6-9) for 30 min (lanes 2 and 6),1h(lanes 3 and 7),2h(lanes 4 and 8),and 4h(lanes 5 and 9) at 25 °C. M, molecular mass marker; lane 1, protein without glutaraldehyde. The cross-linked products were electrophoresed on a 15% SDS-polyacrylamide gel and silver-stained. The migration of monomer, dimer, trimer, and tetramer are indicated on the right.

 



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 5.
Coiled 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.

 
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 (18) or L protein (36). These results clearly suggest that RPC forms a hetero-oligomer with wild-type P protein and thereby inhibits the function of the latter, because RPC lacks the other required functional domains. A similar result was observed when the PPRV P multimerization domain (amino acids 264-386) was used in the minigenome replication/transcription system in place of RPC (Fig. 6b). These results indicate that multimerization of P protein is essential for its function, and the multimerization domain is highly conserved between these two morbilliviruses. To further confirm this conservation of multimerization domain between the two viruses, the wild-type RPV P protein with the histidine tag at its N terminus was coexpressed with the untagged PPRV P multimerization domain in E. coli and purified by nickel affinity chromatography. As shown in Fig. 7, untagged PPRV P multimerization domain is specifically co-purified with wild-type RPV P. The untagged PPRV P multimerization domain expressed alone could not be purified under similar conditions (data not shown). These results suggest that the multimerization domain of PPRV P can interact with RPV P signifying the conserved structure of this domain between the two viruses.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 6.
In vivo replication/transcription assay. a, coiled coil region of RPV P (RPC) or b, multimerization domain of PPRV P (PPMD) was co-expressed with RPV P, and the amount of CAT expressed was estimated by ELISA as described under "Experimental Procedures." Lane 1, A549 cells; lane 2,1 µg of pRP6 without any inhibitory plasmid; lane 3, 1 µg of pRP6 with 2 µg of pET33b(+) plasmids; lanes 4, 5, and 6, 1 µg of pRP6 with 0.5 µg, 1 µg, and 2 µg of RPC or PPMD plasmids, respectively.

 



View larger version (68K):
[in this window]
[in a new window]
  		FIG. 7.
Interaction of RPV P with the multimerization domain of PPRV P. His-tagged RPV P was co-expressed with untagged PPRV P multimerization domain in E. coli BL21(DE3) and was purified using a Ni-NTA agarose column. ~50 ng of pRP6 plasmid DNA and 100 ng of pRPC plasmid were used for coexpression. Lane 1, co-purification of PPMD (No tag) with pRP6 (His tagged) indicating specific interactions between them. Lane 2, molecular mass markers (kDa) indicated by arrows.

 
Prediction of Secondary Structures and Coiled Coil Regions of RPV PMD—The secondary structure prediction analysis using sequences that are closely related to the P protein showed that the multimerization domain is predominantly helical. In particular, the region from positions 316 to 355 is strongly predicted to be {alpha}-helical with a break of 4 residues around position 345. A few shorter segments of {alpha}-helix were also predicted in the beginning of the multimerization domain. Prediction of a long {alpha}-helical region is consistent with the fact that it could form a coiled coil structure.

The amino acid sequence of the multimerization domain was also subjected to the prediction of coiled coil regions. The regions from about 315 to 365 and from 310 to 375 are predicted as coiled coil regions. The result is shown in Fig. 8. It can be seen that the probability of a coiled coil structure for the region of amino acids 340-360 is about 0.7 if the window size used in the program is 14. However, the estimated probability of coiled coil formation for other window sizes is suggested to be low (of the order of 0.2-0.4) probably because of the fact that these procedures do not consider the possibility of a four-stranded coiled coil. When the residues of RPV PMD in the heptad repeat positions (a-g) were analyzed, most of the nonpolar residues at a and d positions remained conserved. Although there were some drastic substitutions from nonpolar to polar residues at those positions, the interactions were maintained by compensatory changes, thereby maintaining the coiled coil structure. Such nonpolar to polar residue substitutions are also seen in many other coiled coil structures (37).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 8.
The predicted coiled coil region of multimerization domain of RPV P. The probability for different window size is indicated by different symbols as in the inset. Amino acid positions 0-123 in the diagram correspond to 266-388 of the P protein. The program by Lupas et al. (29) was used.

 
Structural Features of RPV PMD—The sequence identity between the multimerization domains of RPV P and SeV P is 14.3%. However if the similarity between the aligned residues in the two proteins are scored, the percent sequence similarity is 55.8%. This represents a low level of similarity, but appears convincing based on the compatibility of various residues in RPV P protein with the structural features in SeV P as well as the potential evolutionary relationship. Based on the comparative sequence analysis, it is very likely that the overall fold of the RPV PMD is similar to that of the crystal structure of SeV P. However, as the sequence identity (14.3%) is low, the details of the structures of these two proteins are likely to differ. The potential roles of various residues in the P protein in rendering stability to the coiled coil structure has also been investigated by generating a low resolution model using the crystal structure of SeV P as the basis.

The crystal structure of SeV PMD shows a tetrameric elongated structure. The N-terminal globular structure (approximately first 50 residues from each subunit) is followed by a parallel long coiled coil structure (21). The interaction between the protomers is present both in the small globular region as well as in the coiled coil region. The three-dimensional model for the amino acid sequence of RPV PMD protein generated using the COMPOSER suite of programs based on the alignment with SeV P is shown in Fig. 9.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 9.
Three-dimensional model of multimerization domain of RPV P. The amino acid sequence of the multimerization domain of RPV P was aligned with that of the sendai virus P, and the three-dimensional model was generated using comparative modeling. Four different strands of the tetramer are shown in four different colors in which side chains of residues involved in coiled coil interaction are indicated by spheres. The program SETOR has been used to produce this figure (38).

 
A detailed examination of the model using interactive graphics suggests that the sequence of the multimerization domain of the RPV P protein could be comfortably accommodated in the fold of the SeV P multimerization domain despite a low sequence similarity between these proteins. Most of the apolar residues are buried in the structure, and polar residues are generally exposed. Many of the apparent drastic substitutions in the multimerization domain of RPV P protein compared with SeV PMD are accommodated by stabilizing interactions, which are different in nature compared with the observations in the crystal structure of SeV PMD.

Fig. 10 shows the amino acid sequences of SeV PMD and the multimerization domain of the RPV P protein with the structural environment at every residue position of SeV PMD shown for both the tetrameric state (cryt) and for a monomeric state (crym). The comparison of these two states show that the four-stranded coiled coil structural model of RPV PMD is largely stabilized by a series of interactions between the subunits involving short apolar side chains and a series of ionic pairs formed by oppositely charged amino acid side chains. This feature is also usually seen in the two- and three-stranded coiled coil structures (37). Many of the side chains that are involved in key interactions across the protomers of the tetramer model are shown in Fig. 9. The residues of SeV PMD that are exposed in the monomeric form, but get buried in the native tetramer form are given with the equivalent residue of the RPV PMD protein in the brackets (Fig. 10): Met-328 (Leu), Leu-332 (Ser), Val-333 (Thr), Ser-351 (Ser), Phe-354 (Ser), Ala-355 (Gln), Ala-358 (Ile), Leu-359 (Glu), Cys-372 (Ile), Gly-373 (Gln), Leu-374 (Asp), Leu-376 (Lys), Ser-377 (Thr), Val-386 (Gln), Leu-393 (Leu), Ile-396 (Leu), Val-400 (Lys), Phe-403 (Ile), Tyr-407 (Lys), Gln-414 (Asn), and Leu-425 (Ser). This shows that in addition to the coiled coil region, the residues in the globular domain participate in intersubunit interactions. Some of the residues involved in intersubunit interactions are either conserved between the two proteins or substituted by another residue of similar chemical characteristics. However in a number of positions, the equivalent residues from the two proteins are significantly different.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 10.
Sequence alignment of the multimerization domain of RPV P (mod) with that of sendai virus P based on structural features. Structural features at the residue positions are shown for the protomeric state (crym) and in the tetrameric state (cryt) of the SeV PMD crystal structure. The program JOY has been used to produce this figure (39). Uppercase, solvent inaccessible; lower-case, solvent accessible; italic, positive {varphi}; breve , cis-peptide; tilde, hydrogen bond to other side chain; bold, hydrogen bond to main chain amide; underline, hydrogen bond to main chain carbonyl; cedilla, ç, disulfide bond.

 
Val-386 of SeV PMD is replaced by Gln in the RPV PMD. In the modeled structure Gln residues from the adjacent strands are oriented in such a way that a hydrogen-bonding network connecting the side chains is possible, contributing toward the stability of the four-stranded coiled coil structure. Another residue, Val-400, in the coiled coil region of SeV P is replaced by the lysine residue in the RPV P protein. Interestingly, in the model, the side chain of this lysine residue is hydrogen-bonded to the side chain of a glutamate residue from an adjacent strand, which is equivalent to Ser-402 of SeV P. Thus a cyclic network of salt bridges is predicted to stabilize the coiled coil structure. Similarly, the lysine residue of the RPV P protein that is equivalent to Tyr-407 of SeV PMD is hydrogen-bonded, through its side chain, with the side chain of an Asp in an adjacent strand. This Asp replaces Arg-404 of the crystal structure, and the net result of these two residue changes is yet another cyclic network of salt bridges linking adjacent strands. Thus, here is another example of apparently drastic amino acid substitutions (Tyr-407 -> Lys and Arg-404 -> Asp) resulting in the stabilizing interactions in the coiled coil structure. It is generally known that interactions between oppositely charged residues stabilize coiled coil structures (21). Leu-425 of the crystal structure occurs toward the end of the coiled coil structure and is replaced by Ser in the RPV P protein. In the model, the side chain -OH group of this Ser is hydrogen-bonded to the main chain carbonyl in the relative vicinity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
One of the long standing controversies is the exact oligomerization status of the P protein of negative-stranded RNA viruses. Studies on a number of viruses ranging from rhabdovirus to paramyxovirus report a different oligomeric status of P protein including dimer, trimer, and tetramer (9,11-13,17). By gel filtration, DLS, and chemical cross-linking analyses, we have shown that the RPV P protein exists as a tetramer in solution, and the tetramerization is through the coiled coil region of the C-terminal domain. Although earlier work on P proteins from SeV, NDV, and MuV suggested that P could be a trimer, recent structural analyses using various biophysical methods and crystallographic study reveal that SeV P is a tetramer (13, 21). Though VSV P protein is reported to be a trimer, it is also suggested to form a tetramer (9, 11). It might be noted that the study with rabies virus (a rhabdovirus) P also does not exclude the possibility of tetramer formation (12). All these results lead us to conclude that P proteins from this group of RNA viruses can exist as a tetramer.

P proteins exhibit differences in terms of their requirement of phosphorylation for multimerization. While the P protein of VSV and chandipura virus (ChP) (rhabdovirus) require phosphorylation to facilitate its oligomerization, P proteins from paramyxoviruses such as SeV, NDV, MuV, and MV have been shown to oligomerize independent of phosphorylation (7, 9, 17, 19). Moreover unlike VSV P and ChV P, rabies virus (another rhabdovirus) P protein can form an oligomer in the absence of any phosphorylation (12). The RPV P protein used in the present work is unphosphorylated as it was expressed in bacteria and could form a stable tetramer. These results suggest that multimerization of P protein is independent of phosphorylation except in the case of VSV and ChP. Notably the study with VSV P also suggests the presence of a fraction of oligomeric population in the absence of any phosphorylation, and phosphorylation is only involved in the shift of equilibrium toward the multimer formation. Further at high concentration, the unphosphorylated VSV P exists predominately as a multimer (16). Considering these results, it can be generalized that P proteins of mononegaloviruses exist as a multimer, possibly as a tetramer, whose oligomeric form is independent of any phosphorylation.

P proteins of mononegaloviruses also vary with respect to the role of phosphorylation on their biological functions. In SeV, phosphorylation of P has been shown to be dispensable for transcription and replication functions and a similar possibility is suggested for human parainfluenza virus 1 (hPIV1) also (1). Although phosphorylation of P has been reported to be essential for its activity in VSV and RSV, more recently it has been shown that phosphorylation of HRSV P protein is not essential for transcription and replication (40-42). Also VSV P when used in high concentrations is able to bring about transcriptional activity without requiring any phosphorylation (43). This activity of VSV P could be caused by the presence of some oligomers at high concentrations (16). From these studies, it appears that oligomerization and not the phosphorylation of P protein of mononegalovirales is essential for its transcription and replication function. Using a minigenome in vivo replication/transcription assay, we have demonstrated that disruption of RPV P oligomerization inhibits P protein function almost completely. Although a phosphorylation-null mutant of RPV P has been shown to be inactive in an in vivo replication-transcription assay (22), this could be either caused by an inactive conformation of the mutant (substitution of serine to alanine in three places) or a possible requirement of phosphorylation for an unidentified regulatory role of P protein. We hypothesize that phosphorylation, which is dispensable for transcription and replication, may play an important regulatory role particularly during the initial period of viral infection when the concentration of P protein is very low.

The functional inhibition and interaction studies with the PPRV multimerization domain and RPV P protein strongly suggest that this domain is highly conserved between the two morbilliviruses. We add that this functional conservation may be true for all members of the Paramyxoviridae family, based on in-depth sequence comparison between RPV P and SeV P whose crystal structure is known. Although the sequence identity between these two proteins is insignificant, the structural similarity is likely to be very high. Some of the interacting residues are drastically different (nonpolar residues to polar residues substitution and vice versa) between these two viruses but the four-stranded coiled coil structure is maintained by compensatory changes elsewhere in the sequence. These suggest an evolutionary significance of this domain to perform a similar function among all the viruses of this family. Thus, the model further substantiates a common mechanism of P protein function among various negative sense RNA viruses. Although there is no evidence about the involvement of coiled coil structure in multimerization of rhabdovirus P proteins, there is a predicted coiled coil motif at its N terminus. This coiled coil motif might be responsible for multimerization involving possible tetramerization of P proteins among rhabdoviruses. Recently, with rabies virus P protein, it has been shown that the coiled coil motif at the N-terminal domain is not involved in P protein multimerization and multimerization domain resides at the C-terminal domain, which also harbors the predicted coiled coil motif (12). However, in other rhabdoviruses like VSV, there is no predicted coiled coil motif at the C-terminal domain. This could mean that VSV P either oligomerizes through the N-terminal coiled coil motif or through the C-terminal domain which might harbor a coiled coil motif not detectable by prediction programs. Although further experiments are required to understand the multimerization of VSV P, our results on RPV P along with the results from other investigators discussed above suggest that there is a common mechanism for P protein function that acts as a multimer, possibly as a tetramer, and multimerization is independent of phosphorylation.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ A senior research fellow of the University Grant Commission, Government of India. Back

|| Supported by the Senior Research Fellowship program for Biomedical Research by the Wellcome Trust, London. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Tel.: 91-80-23600139/22932702; Fax: 91-80-23602697; E-mail: shaila{at}mcbl.iisc.ernet.in.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank the Department of Biotechnology, Government of India for infrastructural facilities.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Curran, J., and Kolakofsky, D. (1999) Adv. Virus Res. 54, 403-422[Medline] [Order article via Infotrieve]
  2. Sedlmeier, R., and Neubert, W. J. (1998) Adv. Virus Res. 50, 101-139[Medline] [Order article via Infotrieve]
  3. Lamb, R. A., and Kolakofsky, D. (1996) in Fields Virology (3rd Ed.) (Fields, B. N., Knipe, D. M., and Howley, P. M., eds) pp. 1177-1204, Lippincott-Raven Press, New York
  4. Gao, Y., and Lenard, J. (1995) J. Virol. 69, 7718-7723[Abstract]
  5. Curran, J. (1996) Virology 221, 130-140[CrossRef][Medline] [Order article via Infotrieve]
  6. Curran, J., Marq, J. B., and Kolakofsky, D. (1995) J. Virol. 69, 849-855[Abstract]
  7. Pattnaik, A. K., Hwang, L., Li, T., Englund, N., Nathur, M., Das, T., and Banerjee, A. K. (1997) J. Virol. 71, 8167-8175[Abstract]
  8. Dupuy, L. C., Dobson, S., Bitko, V., and Barik, S. (1999) J. Virol. 73, 8384-8392[Abstract/Free Full Text]
  9. Gao, Y., and Lenard, J. (1995) EMBO J. 14, 1240-1247[Medline] [Order article via Infotrieve]
  10. Barik, S., and Banerjee, A. K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6570-6574[Abstract/Free Full Text]
  11. Gao, Y., Greenfield, N. J., Cleverley, D. Z., and Lenard, J. (1996) Biochemistry 35, 14569-14573[CrossRef][Medline] [Order article via Infotrieve]
  12. Gigant, B., Iseni, F., Gaudin, Y., Knossow, M., and Blondel, D. (2000) J. Gen. Virol. 81, 1757-1761[Abstract/Free Full Text]
  13. Tarbouriech, N., Curran, J., Ebel, C., Ruigrok, R. W., and Burmeister, W. P. (2000) Virology 266, 99-109[CrossRef][Medline] [Order article via Infotrieve]
  14. Asenjo, A., and Villanueva, N. (2000) FEBS Lett. 467, 279-284.[CrossRef][Medline] [Order article via Infotrieve]
  15. Banerjee, A. K. (1987) Cell 48, 363-364[CrossRef][Medline] [Order article via Infotrieve]
  16. Das, T., Gupta, A. K., Sims, P. W., Gelfand, C. A., Jentoft, J. E., and Banerjee, A. K. (1995) J. Biol. Chem. 270, 24100-24107[Abstract/Free Full Text]
  17. Chattopadhyay, D., Raha, T., and Chattopadhyay, D. (1997) Virology 239, 11-19[CrossRef][Medline] [Order article via Infotrieve]
  18. Shaji, D., and Shaila, M. S. (1999) Virology 258, 415-424[CrossRef][Medline] [Order article via Infotrieve]
  19. Harty, R. N., and Palese, P. (1995) J. Gen. Virol. 76, 2863-2867[Abstract/Free Full Text]
  20. Chaoudhary, S. K., Malur, A. G., Huo, Y., De, B. P., and Banerjee, A. K. (2002) Virology 302, 373-382[CrossRef][Medline] [Order article via Infotrieve]
  21. Tarbouriech, N., Curran, J., Ruigrok, R. W., and Burmeister, W. P. (2000) Nat. Struct. Biol. 7, 777-781[CrossRef][Medline] [Order article via Infotrieve]
  22. Kaushik, R., and Shaila, M. S. (2004) J. Gen. Virol. 85, 687-691[Abstract/Free Full Text]
  23. Baron, M. D., and Barrett, T. (1997) J. Virol. 71, 1265-1271[Abstract]
  24. Greenfield, N., and Fasman, G. D. (1969) Biochemistry 8, 4108-4116[CrossRef][Medline] [Order article via Infotrieve]
  25. Wu, C. S. C., Ikeda, K., and Yang, J. T. (1981) Biochemistry 20, 566-570[CrossRef][Medline] [Order article via Infotrieve]
  26. Chen, Y. H., Yang, J. T., and Chau, K. H. (1974) Biochemistry 13, 3350-3359[CrossRef][Medline] [Order article via Infotrieve]
  27. Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584-599[CrossRef][Medline] [Order article via Infotrieve]
  28. Rost, B. (1996) Methods Enzymol. 266, 525-539[CrossRef][Medline] [Order article via Infotrieve]
  29. Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164[Free Full Text]
  30. Berger, B., Wilson, D. B., Wolf, E., Tonchev, T., Milla, M., and Kim, P. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8259-8263[Abstract/Free Full Text]
  31. Wolf, P., Kim, P. S., and Berger, B. (1997) Protein Sci. 6, 1179-1189[Medline] [Order article via Infotrieve]
  32. Srinivasan, N., and Blundell, T. L. (1993) Protein Eng. 6, 501-512[Abstract/Free Full Text]
  33. Weiner, S. I., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., Profeta, S., and Weiner, P. (1984) J. Am. Chem. Soc. 106, 765-784[CrossRef]
  34. Karlin, D., Longhi, S., Receveur, V., and Canard, B. (2002) Virology 296, 251-262[CrossRef][Medline] [Order article via Infotrieve]
  35. Lau, S. Y., Taneja, A. K., and Hodges, R. S. (1984) J. Biol. Chem. 259, 13253-13261[Abstract/Free Full Text]
  36. Chattopadhyay, A., and Shaila, M. S. (2004) Virus Genes 28, 169-178[CrossRef][Medline] [Order article via Infotrieve]
  37. Akey, D. L., Malashkevich, V. N., and Kim, P. S. (2001) Biochemistry 40, 6352-6360[CrossRef][Medline] [Order article via Infotrieve]
  38. Evans, S. V. (1993) J. Mol. Graphics 11, 134-138[CrossRef][Medline] [Order article via Infotrieve]
  39. Mizuguchi, K., Deane, C. M., Blundell, T. L., Johnson, M. S., and Overington, J. P. (1998) Bioinformatics 14, 617-623[Abstract/Free Full Text]
  40. Takacs, A. M., Barik, S., Das, T., and Banerjee, A. K. (1992) J. Virol. 66, 5842-5848[Abstract/Free Full Text]
  41. Barik, S., McLean, T., and Dupuy, L. C. (1995) Virology 213, 405-412[CrossRef][Medline] [Order article via Infotrieve]
  42. Villanueva, N., Hardy, R., Asenjo, A., Yu, Q., and Wertz, G. (2000) J. Gen. Virol. 81, 129-133[Abstract/Free Full Text]
  43. Spadafora, D., Canter, D. M., Jackson, R. L., and Perrault, J. (1996) J. Virol. 70, 4538-4548[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
M. Chen, T. Ogino, and A. K. Banerjee
Mapping and Functional Role of the Self-Association Domain of Vesicular Stomatitis Virus Phosphoprotein
J. Virol., October 1, 2006; 80(19): 9511 - 9518.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
M. T. Llorente, B. Garcia-Barreno, M. Calero, E. Camafeita, J. A. Lopez, S. Longhi, F. Ferron, P. F. Varela, and J. A. Melero
Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an {alpha}-helical domain involved in oligomerization
J. Gen. Virol., January 1, 2006; 87(1): 159 - 169.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
P. Moller, N. Pariente, H.-D. Klenk, and S. Becker
Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription
J. Virol., December 1, 2005; 79(23): 14876 - 14886.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/22/23606    most recent
M400673200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rahaman, A.
Right arrow Articles by Subbarao Shaila, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rahaman, A.
Right arrow Articles by Subbarao Shaila, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement