1 Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France
2 Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France
Correspondence
Jean-François Eléouët
eleouet{at}jouy.inra.fr
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ABSTRACT |
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INTRODUCTION |
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The mechanisms of specific recognition and encapsidation of the viral genomic RNA by the nucleocapsid protein are not well understood. The encapsidation signals are contained in the leader (Le) and Trailer (Tr) regions, which are very similar and situated at the 3' ends of the genomic and antigenomic RNAs, respectively (Collins et al., 2001). When expressed alone in insect cells, the RSV N proteins form nucleocapsid-like structures, due to non-specific binding to cellular RNA (Meric et al., 1994
; Bhella et al., 2002
). Likewise, when expressed alone in E. coli, the N protein binds RNA in a non-specific manner (Murphy et al., 2003
). Thus, the nucleocapsid proteins of these viruses have intrinsic RNA-binding properties that might be controlled or modulated by other factors. For the paramyxoviruses Sendai virus and measles virus, it has been proposed that prior to nucleocapsid assembly, N associates with the P protein, acting as a chaperone for N and preventing non-specific binding to cellular RNAs (Curran et al., 1995
; Spehner et al., 1997
). Co-expression of the rabies virus N and P proteins in insect cells allowed the purification of a NP soluble complex that does not contain RNA (Mavrakis et al., 2003
). Thus, the N protein might be targeted specifically to the Le and Tr nascent chains as an NP complex.
The RSV P protein can interact with L (Khattar et al., 2001a), N (Garcia et al., 1993
; Samal et al., 1993
; Garcia-Barreno et al., 1996
; Mallipeddi et al., 1996
; Slack & Easton, 1998
; Khattar et al., 2001b
), M2-1 (Mason et al., 2003
) and with itself, forming homo-tetramers (Asenjo & Villanueva, 2000
). It is a 241 aa protein that is phosphorylated essentially by cellular casein kinase II on several serine residues located in the middle (positions 116, 117 and 119) and in the C terminus (positions 232 and 237) of the molecule (Navarro et al., 1991
; Mazumder et al., 1994
; Villanueva et al., 1994
; Sanchez-Seco et al., 1995
; Dupuy et al., 1999
). The paramyxovirus P protein is thought to exert at least two different functions: (i) to position the L protein on the NRNA template, (ii) to serve as a chaperone for newly synthesized N, named N0 (Curran et al., 1995
). For RSV, the nature of these complexes remains unclear, and in particular the stoichiometry of LP and NP complexes is still unknown.
There is still debate about the oligomeric status of P and the role of phosphorylation in P oligomerization. RSV P protein transiently expressed in Hep-2 cells is a tetramer (Asenjo & Villanueva, 2000). Mutagenesis of serine residues has resulted in modification of P oligomerization and/or function, suggesting that P phosphorylation is necessary for its oligomerization and transcriptional activity (Barik et al., 1995
; Asenjo & Villanueva, 2000
; Villanueva et al., 2000
). However, structural modifications of the P protein, rather than loss of phosphorylation of these residues, could explain the apparent requirement for phosphorylation (Lu et al., 2002
). It is not known whether association of RSV P with N or with L depends on its oligomeric state and phosphorylation.
In the present work, we have expressed the RSV N and P proteins in E. coli to study PP and PN complexes. The ability of unphosphorylated P to form oligomers and to associate with N was investigated, and the P oligomerization domain was mapped to a short amino acid stretch in the central part of P.
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METHODS |
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Expression and purification of GST-fusion proteins.
E. coli BL21(DE3) cells transformed with pGEX plasmids were grown at 37 °C for 8 h in 125 ml of LuriaBertani (LB) medium containing 100 µg ampicillin ml1. The same volume of LB was then added and protein expression was induced by adding 80 µg IPTG ml1 to the medium. Bacteria were grown at 25 °C and harvested by centrifugation 15 h after induction. Bacterial pellets were resuspended in 10 ml of lysis buffer (50 mM Tris/HCl pH 7·8, 60 mM NaCl, 1 mM EDTA, 2 mM DTT, 0·2 % Triton X-100, 10 mM MgSO4, 1 mM CaCl2) supplemented with complete protease inhibitor cocktail (Roche) and incubated for 1 h on ice. RQ1 RNase-free DNase (Promega) was added to the lysates (final concentration 1 U ml1) and incubated at 25 °C for 30 min. Lysates were spun at 10 000 g for 30 min at 4 °C. GlutathioneSepharose 4B beads (Pharmacia) were added to the clarified supernatants (100 µl of beads for 250 ml of induced bacteria culture) and rotated at 4 °C for 15 h. Beads were washed three times with lysis buffer and then stored at 4 °C.
Cultures of recombinant E. coli BL21 harbouring the two plasmids pGEX and pET were grown in 125 ml of LB containing ampicillin (100 µg ml1) and kanamycin (50 µg ml1), and harvested as described above.
To separate RSV recombinant proteins from GST, beads were incubated with biotinylated thrombin as described by the manufacturer (Novagen). Biotinylated thrombin was removed by the Thrombin Cleavage Capture kit as described by the manufacturer (Novagen).
In vitro translation of N and P proteins.
[35S]Methionine-labelled N and P proteins were translated in vitro using the TNT kit (Promega). Reactions were carried out using 1 µg of pET-N or pET-P plasmid in a 50 µl transcription/translation reaction containing 2 µl of translation grade [35S]methionine [1000 µCi ml1 (37 MBq ml1); ICN].
GST pull-down assays.
Mock- or RSV-infected Hep-2 cells [24 h post infection (p.i.)] grown in 75 cm2 dishes were incubated with 50 µl of Promix [1000 µCi ml1 (37 MBq ml1); ICN] in 5 ml of culture medium. Cells were incubated overnight at 37 °C, washed twice in 1x PBS and lysed for 1 h at 4 °C in 4 ml of lysis buffer containing 10 mM Tris/HCl pH 7·2, 350 mM NaCl, 0·5 mM EGTA, 0·5 mM EDTA, 1 % Triton X-100, 1 mM DTT, 20 % glycerol, supplemented with complete protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation at 10 000 g for 15 min at 4 °C. Ten microlitres of the prepared glutathioneSepharose 4B beads bound to GST-fusion proteins were mixed with cell lysates (250 µl) and rotated end-over-end for 4 h at 4 °C. For assays employing in vitro translated proteins (IVT), beads were incubated with 5 µl of 35S-labelled IVT reactions in 50 µl of bacteria lysis buffer described above. The beads were collected by centrifugation for 5 min at 500 g at 4 °C and washed three times with lysis buffer. Pull-down eluates were heat denatured in Laemmli buffer and run on 12 % SDS-polyacrylamide gels, fixed and stained in solution containing 20 % ethanol, 10 % acetic acid and 0·25 % Coomassie brilliant blue R250, washed in fixing solution (20 % ethanol and 10 % acetic acid), dried and exposed by autoradiography.
Generation of antisera.
Polyclonal antisera were prepared by immunizing rabbits three times at 2 week intervals using purified GST-fusion proteins (100 µg) for each immunization. The first and second immunizations were administered subcutaneously in 1 ml Freund's complete and Freund's incomplete adjuvant (Difco), respectively. The third immunization was done intramuscularly in Freund's incomplete adjuvant. Animals were bled 10 days after the third immunization.
Mass spectrometry analysis.
A 10 µl volume of recombinant protein was used (concentration 1·5 mg ml1). The sample was desalted on ZipTip C4 (Millipore) and eluted with 1 µl of 70 % (v/v) acetonitrile/0·3 % (v/v) trifluoroacetic acid. The sample was directly spotted onto the MALDI plate and dried at room temperature; 0·5 µl of 2,5-dihydroxybenzoic acid (DHB; 10 mg ml1) in water was added. Mass spectra were acquired on a Voyager-DE-STR time-of-flight mass spectrometer (Applied Biosystems) equipped with a nitrogen laser emitting at =337 nm (Laser Science). The accelerating voltage used was 25 kV. All spectra were recorded in the positive reflector mode with a delayed extraction of 1700 ns and a 94 % grid voltage. The spectra were calibrated using an external calibration: cytochrome c, [M+H]+=12362·0 Da; trypsin [M+H]+=23464·5 Da.
Amino acid sequencing.
Electrophoretic samples were transferred onto PVDF membrane by passive absorption. The bands of interest were excised, dried in a Speed-vac for 30 min, and the gel pieces were rehydrated in the initial volume of excised band (2 % SDS, 0·2 M Tris/HCl pH 8·5) for 1 h. After rehydration, 5 vols of water were added and then a piece of prewet (methanol) 4x4 mm PVDF membrane (Problott Applied Biosystems) was added to the solution. At the end of this transfer time (2 days at room temperature), the membrane was washed five times with 1 ml 10 % methanol with vortexing. N-terminal Edman sequencing was performed on an Applied Biosystems Procise 494HT with reagents and methods recommended by the manufacturer.
Cross-linking assays.
GST-fusion proteins bound to glutathioneSepharose beads were extensively washed with 1x PBS and cleaved with thrombin as described by the manufacturer (Novagen). The resulting proteins were incubated for 1 h at room temperature with increasing concentrations of freshly diluted glutaraldehyde in 1x PBS. Reactions were stopped by the addition of lysine at 200 mM for 30 min at room temperature.
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RESULTS |
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To determine whether some other oligomeric forms of P*, such as trimers, were present in the samples but in lower amounts, the P* protein was cross-linked with glutaraldehyde at a final concentration of 0·025 % and analysed by Western blot using an anti-P rabbit polyclonal serum, as this technique is more sensitive than Coomassie blue staining. The strength of PP interactions was estimated by incubating the P* protein with increasing concentrations from 0 to 1 M of NaCl. As shown in Fig. 4(D), only two major complexes migrating with apparent masses of 70 and 140 kDa were again detected. These results suggest that the P complex might be constituted of two dimers that assemble into tetramers. Formation or stability of P dimers and tetramers were not inhibited by high concentrations of NaCl (1 M).
Prediction of the coiled-coil oligomerization domain of P
RSV P protein sequence was submitted to a coiled-coil prediction program that determined two potential regions situated approximately between aa 120150 and 175215, respectively (Fig. 5A). Generally, coiled-coils are alpha-helices which have hydrophobic interfaces and a hydrophilic exterior. Every first and fourth amino acid of a 7 residue repeat in the helix is hydrophobic. As shown in Fig. 5(B)
, the first predicted domain (aa 129152) presents a cyclic (3 or 4 aa) hydrophobic residues repetition. This was not found in the second computer-predicted region (175215). Interestingly, sequence alignment of phosphoproteins of several pneumoviruses revealed that the putative first coiled-coil domain is part of a highly conserved domain (Fig. 5C
). This conserved domain is situated in the central part of P proteins and seems to be limited in C terminus by a conserved GP (positions 158159) that is incompatible with an alpha-helix structure. The other parts of the molecule show low sequence similarities between these pneumoviruses.
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Bacterial RNA is co-purified with GST-N, but not with GST-N+His-P or GST-P+His-N complexes
When the GST-N fusion protein was cut by thrombin or eluted from glutathioneSepharose by 50 mM glutathione, the protein was essentially present in the pellet after centrifugation for 1 min at 10 000 g, indicating that it was not soluble, possibly because of the association of N with bacterial RNA. In order to determine whether RNA was present in the preparations, 10 µg of GST-N, GST-N+His-P or GST-P+His-N purified protein complexes adsorbed on glutathioneSepharose beads was heat denatured for 5 min at 100 °C, and run on a 2 % agarose gel stained with ethidium bromide. As shown in Fig. 7(D), presence of RNA was revealed for the GST-N protein, whereas only trace amounts of RNA were visible for GST-N+His-P and GST-P+His-N. The presence of bacterial RNA was never detected for GST or GST-P by this method (data not shown). These results showed that unspecific binding of N0 to bacterial RNA is prevented by the presence of P within the same bacteria, allowing soluble N0P complex formation.
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DISCUSSION |
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We investigated the role of phosphorylation in P oligomerization, firstly without modifying the amino acid sequence of the protein. In infected cells, about 80 % of P phosphorylation involves Ser-232, and the remainder involves Ser-116, -117, -119 and -237 (Barik et al., 1995; Sanchez-Seco et al., 1995
). Mazumder et al. (1994)
reported that P purified from bacteria was not phosphorylated and chromatographed with an apparent size of 120150 kDa, suggesting that the P protein behaved as a trimer or a tetramer. Asenjo & Villanueva (2000)
reported that recombinant RSV P protein purified from E. coli was unable to oligomerize. However, mutation of the five phosphorylated serines on RSV P protein did not abolish oligomer formation in Hep-2 cells. Thus, it was postulated that a transitory phosphorylation of P protein could occur and play a role in oligomerization. In this report, we clearly show that when expressed in bacteria the RSV P recombinant protein is able to oligomerize. Mass spectrometry analysis confirmed that recombinant P purified from E. coli was not phosphorylated. Gel filtration studies suggested that the purified P protein was homogeneous, with an apparent mass compatible with P being a tetramer. Chemical cross-linking experiments and Western blotting analysis of the cross-linked products confirmed the presence of tetramers. At lower glutaraldehyde concentrations, complexes corresponding to P dimers were also found. Because trimers were not detected, the presence of P dimers might reflect a structural suborganization of the tetramers. As high molecular mass complexes were not observed by gel filtration, these complexes might only be formed during glutaraldehyde cross-linking. These results demonstrate that P phosphorylation is not required for P oligomerization. Furthermore, the P domain comprising aa 120150, which does not contain phosphorylated serine, was still able to oligomerize. Thus, the role of RSV P protein phosphorylation might reside in other function(s). In vitro reconstituted transcription experiments suggested that P phosphorylation is involved in transcriptional activity of RSV P protein (Mazumder & Barik, 1994
; Barik et al., 1995
). Other data, obtained by mutagenesis of serines, suggested that P phosphorylation could play a role in PL stabilization during transcription elongation activity of the viral polymerase (Dupuy et al., 1999
). However, in vitro phosphorylation of P protein did not result in transcriptionally active P protein (Dupuy et al., 1999
). Recently, Lu et al. (2002)
suggested, by using reverse genetics, that P phosphorylation could be involved in virus budding rather than in virus replication. Finally, it was shown that PM2-1 interaction is independent of P phosphorylation (Mason et al., 2003
). All the data, together with our results, indicate that P phosphorylation is not needed for PP, PN and PM2-1 interactions, and that RSV replication occurs without P phosphorylation. Even though PL interactions have not been extensively investigated, we suggest that P phosphorylation could be required in other aspects of the virus-cycle. It could be involved in the formation of frozen polymerase complex before the release of virions from infected cells, or necessary for interaction of the P protein with the Matrix (M) protein, to initiate the formation of new RSV particles.
The use of positive interaction screenings allowed us to map the P oligomerization domain to aa 120150. Comparison between several members of the Paramyxoviridae family showed that the P protein is not well conserved at the amino acid level (not shown). However, sequence alignment restricted to four different members of the Pneumovirus genus revealed that this domain is situated in, and represents the major part of, a well conserved region in the middle of the molecule, the other parts showing low or no sequence similarities. Amino acid sequence comparison with the paramyxovirus Sendai virus phosphoprotein for which the atomic structure has been determined as being a tetramer (Tarbouriech et al., 2000), did not reveal any similarities (data not shown). However, these two domains could have a comparable secondary coiled-coil structure.
The role of P phosphorylation for NP interaction has been investigated by using reverse genetics (Lu et al., 2002). NP interaction was reduced to 40 % when Ser-116, -117, -119 (central region), and -232 and -237 (C-terminal region) were mutated, suggesting that P phosphorylation could be important for NP interactions. Our results showed that N0P complex formation was very efficient in bacteria in the absence of phosphorylation. Thus, the reduced NP interaction observed with recombinant viruses could be due to structural changes caused by serine mutations rather than lack of P phosphorylation.
The nucleocapsid protein of RSV associates with RNA with no sequence specificity when expressed alone, forming nucleocapsid-like structures (Bhella et al., 2002). When GST-N from bacteria was purified the fusion protein was poorly soluble after thrombin cleavage or elution by glutathione, and contained high amounts of RNA. Purified GST-NRNA complexes were not displaced by addition of soluble P* protein (not shown). In order to maintain N in a soluble form (designated N0), we co-expressed N and P proteins in bacteria, and an N0P complex was purified. This complex was soluble and did not contain high amounts of bacterial RNA. The same results were obtained recently with rabies virus N and P proteins co-expressed in insect cells, using recombinant baculoviruses (Mavrakis et al., 2003
). Our experiments confirm that non-specific binding of RSV N protein to RNA is prevented by P, and that binding of N to RNA is strong and probably irreversible. The specificity of RNA-binding of the N0P complex to viral RNA sequences will be investigated in future experiments using this purified N0P complex.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 November 2003;
accepted 22 January 2004.