Human respiratory syncytial virus matrix protein is an RNA-binding protein: binding properties, location and identity of the RNA contact residues

Lorena Rodríguez, Isabel Cuesta, Ana Asenjo and Nieves Villanueva

Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, Majadahonda, Madrid 28220, Spain

Correspondence
Nieves Villanueva
nvilla{at}isciii.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human respiratory syncytial virus (HRSV) matrix (M) protein is a structural internal membrane protein. Here we have shown that, like its orthomyxovirus and rhabdovirus counterparts, it has RNA-binding capacity, as determined by retardation of 32P-labelled riboprobes in gel electrophoresis, cross-linking with UV light and Northern-Western assays. Its binding to RNA was neither sequence-specific nor showed a length requirement, although it had cooperative kinetics with a Kd of 25 nM and probably two different types of RNA-binding sites. After preparative cross-linking of 32P-labelled riboprobes with purified, renatured HRSV Long strain M protein (256 residues), the residues in contact with RNA were located between amino acids 120 and 170, in the central part of the molecule. Lysine (positions 121, 130, 156 and 157) and arginine (position 170) residues located within this region and conserved among pneumovirus M proteins of different origins were found to be essential for RNA contact. M protein expression did not affect the replication and transcription of HRSV RNA analogues in vivo (except when expressed in large amounts), in contrast to the in vitro transcription inhibition described previously. In addition, M protein was found to aggregate into high-molecular-mass oligomers, both in the presence and absence of its RNA-binding activity. The formation of these structures has been related in other viruses to either viral or host-cell RNA metabolism.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human respiratory syncytial virus (HRSV), a paramyxovirus of the genus Pneumovirus, is the main causative agent of pneumonia in infants. In a recent laboratory study, it was also among the four most frequently confirmed causes of pneumonia in adults, together with Streptococcus pneumoniae, influenza virus and Mycoplasma pneumoniae (Zambon et al., 2001).

Studies suggest that vaccines formed from attenuated viruses, humanized neutralizing monoclonal antibodies and specific antiviral compounds are the most suitable tools for controlling HRSV infection (Collins et al., 2001). The determination of viral protein function at the molecular level during the virus growth cycle would be a first step towards improving these reagents. HRSV virions are polymorphic, with a membranous envelope. The viral glycoproteins G, F and SH are embedded in the outer part of the membrane and the inner surface contacts the matrix (M) protein. By analogy with other paramyxoviruses, it is thought that homologous interactions of M protein molecules form a sheet that interacts with the viral ribonucleoprotein (vRNP) and with the cytoplasmic tails of the viral glycoproteins (Peeples, 1991). The interaction of the M protein with the vRNP, which is composed of the viral N protein tightly bound to the vRNA or cRNA to form the nucleocapsids, and the L protein bound to its cofactors, the phosphoproteins P and M2-1, is mediated by the N protein (Ghildyal et al., 2002; Murphy et al., 2003; Ulloa et al., 1998).

The M protein is crucial in extracellular virus particle formation (Teng & Collins, 1998). It results in the formation of extracellular infectious virus or infectious filamentous structures that protrude from infected cell membranes, in the absence of the ecto- and transmembrane domains of the SH, G and F glycoproteins, but in the presence of the F cytoplasmic tail (Oomens et al., 2003). When expressed alone, M protein is able to bind cellular plasma membranes. This binding is stabilized by surface expression of the viral glycoproteins, and its co-localization with the F protein has been described in specific lipid rafts (Henderson et al., 2002).

The M proteins of several enveloped viruses, when expressed in the absence of the remaining viral proteins, promote the formation of membranous vesicles within which the M protein is released to the extracellular medium (Gomez-Puertas et al., 2000; Li et al., 1993; Timmins et al., 2001). Co-expression of viral glycoproteins or nucleocapsids (Coronel et al., 1999; Henderson et al., 2002; Schmitt et al., 2002) increases the formation of these virus-like particles (VLPs). This suggests that the M protein promotes paramyxovirus budding by acting as a bridge between the nucleocapsid and the viral envelope. It was suggested that the M protein binds simultaneously to cellular actin (the main component of cell cytoskeletal microfilaments) and the viral RNPs, facilitating transport of these viral structures to the plasma membrane location into which the mature viral glycoproteins are inserted (Burke et al., 1998; Naito & Matsumoto, 1978). This could explain the presence of actin in purified preparations of various extracellular viral particles (Naito & Matsumoto, 1978; Ott et al., 1996; Ulloa et al., 1998).

The M proteins of influenza A virus, vesicular stomatitis virus (VSV) and HRSV are able to alter the structures of nucleocapsids that are actively synthesized in vitro, rendering them inactive for viral RNA synthesis (De et al., 1982; Ghildyal et al., 2002; Ye et al., 1987). The single-strand RNA-binding capacity of influenza A virus M protein (Wakefield & Brownlee, 1989) suggests that the interaction between nucleocapsids and the M protein is mediated by RNA, but further experiments are needed to confirm this. The Ebola virus M protein has this capacity (Gomis-Ruth et al., 2003). It is suggested that incorporation of the nucleocapsid in extracellular particles in parainfluenza and VSV viruses is mediated by direct N–M protein interactions (Coronel et al., 2001; Kaptur et al., 1991). The M protein may thus bind to viral nucleocapsids through RNA and N protein interactions.

The structural role of the M protein recently became more evident, as it was found that rabies virus M protein regulates the balance of virus transcription and replication (Finke et al., 2003), although it is not known whether it has RNA-binding capacity. In Ebola virus, M protein RNA-binding capacity is related to its ability to produce pore-like structures related to cell or viral RNA metabolism (Gomis-Ruth et al., 2003). Early in infection, HRSV M protein could inhibit host-cell transcription, a fact related to its location in the cell nucleus at early but not late times during infection (Ghildyal et al., 2003). Nuclear localization has also been described for the Sendai virus M protein (Stricker et al., 1994).

Here we report for the first time that the HRSV M protein has RNA-binding capacity and characterize some of its binding properties.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
HEp-2 and CV1 cells, HRSV Long strain and the vaccinia recombinant vTF-3 viruses were as described previously (Villanueva et al., 1991, 2000).

HRSV infection and extracellular virus particle purification.
The experimental conditions followed were those described previously (Villanueva et al., 1994; Ulloa et al., 1998).

Plasmids.
cDNA corresponding to the N, P, M and M2 genes of HRSV Long strain, previously cloned in the pBSV9 expression vector (López et al., 1988), were subcloned in the SmaI site of the plasmid vector pGEM3 as HpaI–StuI fragments. For the N gene, a point mutation was introduced, which converts the N protein into that of the A2 strain. For the M2 gene, two pGEM3 recombinant plasmids were obtained: pGEM3M2 contains the intact cDNA from the M2 gene and pGEM3M2-1 contains a stop codon at nt 712–714 of the M2 gene. Recombinant pGEM3 plasmids containing the cDNA corresponding to the M protein variants were obtained by directed mutagenesis (Higuchi et al., 1988). All HRSV cDNA gene nucleotide sequences were determined by automatic sequencing.

The plasmid pcL, previously named pL (Cuesta et al., 2000), was also used. Plasmids pWT5, pM/SH and pL, whose construction has been previously described (Hardy & Wertz, 1998), were kindly supplied by Dr G. Wertz. They contained sequences from the HRSV A2 strain; pL contained the N1049D point mutation, which decreases transcription termination (Cartee et al., 2003).

Transfection
For protein synthesis.
cDNA of viral M protein and its variants was transiently expressed in the vaccinia-based expression system under the control of the T7 RNA polymerase promoter as described previously (Cuesta et al., 2000). Soluble proteins were prepared from each transfection as described previously (Cuesta et al., 2000). Only M and the VM15, VM16 and VM17 M protein variants were found in both soluble and insoluble forms; the remaining M protein variants were found mainly in insoluble form and were therefore solubilized using SDS-PAGE sample buffer (Cuesta et al., 2000).

For RNA synthesis.
The conditions described by Villanueva et al. (2000) were followed. To assay the different M proteins, the total amount of plasmid DNA for transfection was 12 µg rather than 15 µg in HEp-2 cells growing on 21 cm2 plastic surfaces (equivalent to 4·2 µg on an 8 cm2 surface).

M protein purification.
M protein purification and renaturation procedures were as described for HRSV M2-1 protein (Cuesta et al., 2000).

Preparation of labelled riboprobes.
Plasmids pGEM3N, pGEM3P and pcL were linearized by digestion with restriction enzymes and riboprobes were prepared following previously described protocols (Cuesta et al., 2000). The positive polarity riboprobes used were Pm (1000 nt), Nm (1200 nt), pcL700 (700 nt) and pcL86 (86 nt), and the negative polarity probes were Pv (1000 nt) and Nv (1200 nt).

UV cross-linking assay.
Various amounts of M protein (10–180 ng) were mixed with 1–5 ng 32P-labelled riboprobe (specific activity 30 000 Cerenkov c.p.m. ng-1) and incubated for 1 h at room temperature. The protein was UV-cross-linked to the riboprobe as described previously (Albó et al., 1995). After treatment with 60 µg RNase A ml-1 from bovine pancreas, 12 % SDS-PAGE was carried out, and gels were stained with Coomassie blue and autoradiographed.

Gel retardation assay.
The protein and riboprobes were incubated under the binding conditions described above, after which electrophoresis was carried out at 100 V in a gel containing 3·5 % acrylamide and 0·5x TBE buffer. After fixing with 95 % ethanol, drying and autoradiography, radioactivity in the riboprobe position was quantified using a PhosphorImager.

Northern-Western assay.
Total protein (30–40 µg) from transfection experiments containing similar amounts of M protein variants was separated by SDS-PAGE and blotted onto nitrocellulose. After several washes with 20 mM NaCl, 40 mM Tris/HCl, pH 8·0, proteins were renatured by incubation in the same buffer containing 0·02 % PVP-40, 0·02 % BSA, 0·02 % Ficoll 400 and 0·1 % Triton X-100 for 3 h at room temperature. The 32P-labelled riboprobe was added in the same buffer containing yeast RNA (1 µg ml-1). After a 2 h incubation, the probe was removed and the membrane washed several times in renaturation buffer. The dried membrane was exposed and quantified using a PhosphorImager.

Chemical and enzymic treatment of the M protein.
The cross-linking reaction described above was carried out with 80 ng or 4·8 µg purified M protein and 10 ng or 30 ng of 32P-labelled riboprobe Pv, respectively. After RNase A digestion and acetone precipitation, the labelled M protein was first mixed with 750 ng purified M protein. Mixtures were treated with cyanogen bromide (CNBr) and trypsin (Fontana & Gross, 1986; Wilkinson, 1986), then processed as described previously (Cuesta et al., 2000). The peptides generated were separated by electrophoresis, transferred to an Immobilon membrane and detected by staining and autoradiography. The N-terminal ends were determined by partial sequencing (Cuesta et al., 2000).

Glutaraldehyde cross-linking assays.
The conditions described by Asenjo & Villanueva (2000) were followed using 1·5 mg protein ml-1 from the soluble transfection protein fraction and 0·01 % glutaraldehyde. After 10 % SDS-PAGE and transfer to Immobilon, the M protein and its oligomers were detected by Western blotting using a monospecific rabbit anti-M protein serum.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HRSV M protein binds to RNA
To determine which HRSV Long strain proteins bind RNA, several viral structural proteins were preparatively purified under denaturing conditions and then renatured, including the M protein (Fig. 1A, lanes 1 and 2). Following incubation with 32P-labelled riboprobes, the purified M protein was shown to be cross-linked to the riboprobes following UV treatment (Fig. 1B) and to retard their electrophoretic mobility (Fig. 1C and D). In addition, the M protein was shown to bind RNA in Northern-Western blot assays (see below). This capacity has not been reported for other paramyxovirus M proteins. In electrophoretic mobility retardation assays using 32P-labelled riboprobes, the M protein was found to bind RNA of different lengths (1200, 1000, 700 and 86 nt) with no sequence specificity (vRNA and mRNA from N and P genes and cRNA of 86 and 700 nt) and with cooperative kinetics. An example of these experiments is shown in Fig. 1(C). The apparent dissociation constant (Kd) was approximately 25 nM, ranging from 20–40 nM depending on the nature of the riboprobe used. This value is probably underestimated, since not all M protein molecules assayed would have renatured.



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Fig. 1. RNA-binding properties HRSV M protein. (A) SDS-PAGE analysis of purified and renatured M protein stained with Coomassie blue. Lane V shows the protein composition of purified HRSV viral particles used as the initial material for the isolation and purification of the M protein. Lanes M1 and M2 show the protein composition of approximately 150 and 300 ng of purified M protein preparation, respectively. The electrophoretic mobility of molecular mass markers (Lane MW, shown on right in kDa) and viral proteins (left) are shown. (B) Thirty-five and 70 ng of the M protein preparation shown in (A) (lanes 1and 2) or a similar volume of buffer only (lane 3) were incubated with 2·8 ng 32P-labelled RNA (27 660 Cerenkov c.p.m. ng-1) corresponding to N gene mRNA (1200 nt) under the conditions indicated in the Methods. After UV cross-linking and RNase digestion, samples were analysed by SDS-PAGE and autoradiography. Molecular mass markers (kDa, right) and the position of the viral proteins (left) are shown. (C) Various amounts of purified M protein (as indicated above the insert) were incubated with 5 ng 32P-labelled RNA (10 840 Cerenkov c.p.m. ng-1) corresponding to P gene vRNA (1000 nt) as above, in a final volume of 12·5 µl, then electrophoresed on a 5 % acrylamide gel. Radioactivity at the position corresponding to RNA electrophoretic mobility (lower asterisk) was quantified using a PhosphorImager and calculated as a percentage of that of the riboprobe in the absence of added protein (lane 0), as shown on the graph. The upper asterisk indicates the protein-retarded labelled RNA. (D) Various amounts of 32P-labelled RNA (28 700 Cerenkov c.p.m. ng-1) from the P gene vRNA (1000 nt), as indicated above the insert, were incubated with (+) or without (-) 60 ng purified M protein and analysed as in (C). The ratio between protein-bound (upper asterisk) and free RNA (lower asterisk) (y-axis) was determined and plotted against protein-bound RNA (upper asterisk) (x-axis).

 
In equilibrium binding studies in which a fixed amount of M protein (60 ng) was incubated with increasing amounts of 32P-labelled riboprobe (Pv and pcL700), the M protein exhibited saturable binding at increased RNA concentrations (not shown). When represented by a Scatchard plot (Scatchard, 1949), the data showed two different RNA-binding sites with distinct affinities (Fig. 1D).

Taken together, these results indicate that the HRSV M protein has a dual RNA-binding capacity, a property described for rhabdo-, ortho- and filovirus M proteins, but not previously reported for an M protein of the Paramyxoviridae family.

Mapping of the M protein regions containing the RNA contact residues
Preparative cross-linking was performed and, after RNase digestion, the labelled protein was cleaved with CNBr or trypsin. The peptides generated were separated by electrophoresis, visualized by amido black staining and those labelled with 32P were detected by autoradiography (Fig. 2A). The N-terminal sequence was determined for several peptides generated by both treatments; calculated sizes for peptides and the positions on the M protein of the amino acid residues located at their N termini are summarized in Fig. 2(B). Calculated fragment sizes may be overestimated due to the small peptide sizes and possible bound oligoribonucleotides. The approximate fragment locations are shown on the M molecule, although their exact C-terminal ends are unknown (Fig. 2C). The results indicated that at least some M protein residues in contact with RNA were between positions 96 and 173 or 120 and 173 (since the T1 tryptic peptide end must be near residue positions 120–140, according to its calculated molecular mass). The presence of residues involved in RNA binding around this region or at the C terminus of the M protein cannot be ruled out, since the T2 tryptic fragment could not reach the end of the M protein; in addition, short central or C-terminal labelled fragments may be too small to be isolated by electrophoresis.



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Fig. 2. Mapping of M protein residues in contact with RNA. (A) Purified M protein (80 ng for CNBr cleavage or 2·4 µg for trypsin cleavage) was incubated with 10 or 15 ng 32P-labelled RNA (10 000 or 60 400 Cerenkov c.p.m. ng-1, respectively) corresponding to P gene vRNA (1000 nt) under the conditions indicated in the Methods. After UV cross-linking and RNase digestion, 750 ng purified M protein was added to the first sample and treated with CNBr (8 mg ml-1) in 63 % formic acid at 37 °C for 4 h, as previously described (Fontana & Gross, 1986). The second sample was treated with 1 µg trypsin at 37 °C for 2 h, as previously described (Wilkinson, 1986). The CNBr-treated sample was neutralized with 0·1 M NH4HCO3, pH 8·5. Both samples were analysed by SDS-PAGE containing tricine and 6 M urea and transferred to Immobilon membrane. Peptides were visualized by amido black staining (S); phosphopeptides (32P) were detected after autoradiography. The electrophoretic mobility of molecular mass markers are indicated at the left and right, in addition to the unlabelled and labelled peptides whose N-terminal sequences were determined. (B) The N-terminal sequences determined for the unlabelled (-) and labelled (+) peptides are indicated in addition to their calculated molecular mass. (C) Diagram of the HRSV M protein primary structure, indicating the location of unlabelled (discontinuous lines) and labelled (continuous lines) peptides and the location of the RNA-binding region (bold line). The approximate ends of the peptides are indicated (striped lines).

 
Determination of essential RNA contact residues in the M protein RNA-binding region
To confirm that M protein amino acids between 120 and 173 are those involved in direct interaction with RNA and to delimit the residues essential for RNA binding, we tested the RNA-binding capacity of the M protein and that of various deletion variants in Northern-Western blots using M proteins transiently expressed in the vaccinia-based expression system. cDNA of the HRSV Long strain M gene was expressed in this system and a protein was found with the same electrophoretic mobility as the M protein from purified virions (Fig. 3B, lower panel). M protein deletions covering residues 91–171 and those between positions 186 and 256 are shown in Fig. 3(A).



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Fig. 3. Location of residues between positions 90 and 173 essential for HRSV RNA binding. (A) Diagram of HRSV M protein primary structure and that of the M protein deletion variants. Bold numbers indicate the residues flanking the deletion for each variant. Bold letters indicate the residues added at the C terminus of the VM10 variant during the cloning process. (B) Northern–Western assay (upper panel) of the extract from HEp-2 cells transiently expressing M protein variants in the vTF3-based vaccinia recombinant system. Similar amounts of M protein variants contained in the different extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes. After renaturation and incubation with 32P-labelled riboprobes, the RNA-binding capacity of the variants was visualized by autoradiography. Protein transferred to the membrane was analysed by Western blotting using a monospecific rabbit anti-M serum (lower panel). (C) Quantification of the RNA-binding capacity (averaged values of at least four experiments) of M protein variants taking into account the amount of M protein analysed in each case (specific activity), expressed as a percentage of that of the control M protein. Maximum and minimum values obtained in each case are indicated.

 
In Northern-Western assays, all variants bound long and short as well as positive- and negative-strand RNAs (not shown). Fig. 3(B, upper) shows the results using the pcL86 riboprobe. The RNA-binding capacity of each variant was determined with reference to its mass as determined by Western blotting (Fig. 3B, lower) and expressed as a percentage of that calculated for the whole M protein. Mean values obtained after quantification of results from distinct riboprobes (Fig. 3C) indicated that only M protein variants with deletions of residues 121–130 (VM4), 154–160 (VM7), 155–156/163–165 (VM8), 162–171 (VM9), 209–216 (VM11) and 186–256 (VM10) had decreased binding capacity. These were reduced by approximately 43, 30, 79, 22, 27 and 50 %, respectively, compared with the RNA binding shown by the whole M protein. It thus appears that the M protein binds RNA through residues 121–130 and 154–171, in agreement with the tryptic mapping results. However, binding through residues 209–216 and/or 186–256 could not be ruled out.

Substitution variants were then constructed (Fig. 4A). Residues between 120 and 172 were replaced, concentrating on positions 154–167, since this region contains most of the M protein RNA-binding capacity. Quantification of Northern-Western assay data (Fig. 4B and C) indicated that replacement of lysines by alanines at positions 156 and 157 (VM18) resulted in a 55 % decrease in M protein RNA binding. A further replacement of arginine at position 170 (VM19: VM18 plus R170A) resulted in a further decrease to 23 % of M protein RNA-binding capacity. Residues Y163, L164 and R165 contributed slightly to M protein RNA-binding capacity (VM20 maintained 62 % of M protein RNA binding). When residues 156, 157 and 170 were also replaced (VM22), only 10 % of M protein RNA-binding capacity remained. Thus, some or all of the residues at positions 163, 164 and 165 could be responsible for the binding retained by the VM19 variant. In addition, when residues at positions 156, 157 and 170 were all replaced together with those at positions 121 or 130 (VM23 and VM24), the binding capacity decreased by 92 % in both variants. When substitutions at positions 121 and 130 were added to VM19 (VM25), no binding was detected. It thus appears that residues at positions 121, 130, 156, 157 and 170, but not those at positions 163, 164 and 165 nor among positions 186–256, are responsible for the M protein RNA-binding capacity. Residues K121 and R170 are conserved in pneumovirus M proteins. Residues K156 or 157 and K130 are also conserved, although not at exactly the same positions (Fig. 5).



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Fig. 4. Determination of M protein residues between positions 120 and 173 essential for RNA contact. (A) Diagram of the HRSV M protein showing the primary sequence between residues 121 and 170 and the residues replaced in the M protein variants. (B) Upper panel, Northern–Western assay of the M protein-substituted variants under the conditions outlined in Fig. 3. Lower panel, Western blot of the membrane assayed in the upper panel, developed with a monospecific rabbit anti-M serum. (C) Quantification of the RNA-binding capacity of the substitution variants, analysed as in Fig. 3(C).

 


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Fig. 5. Conservation of residues essential for M protein RNA-binding capacity in different pneumovirus M proteins from distinct hosts. The primary sequence of HRSV Long strain M protein is compared with that of human (subtypes A and B), bovine, ovine, murine, avian (types A, B and C) and metapneumovirus. All sequences except the Long strain and that of ovine origin (accession no. U02470) are taken from Van der Hoogen et al. (2002). Residues essential for the RNA-binding capacity of the HRSV Long strain M protein are labelled with large asterisks (above). Small asterisks (below) show the equivalent residues from pneumovirus M protein of avian origin.

 
Influence of M protein expression on replication and transcription of HRSV RNA analogues
It has been suggested that the Paramyxoviridae family M protein binds to the nucleocapsids that are actively synthesizing RNA, compacting them and stopping their activity. This was shown for HRSV by in vitro analysis (Ghildyal et al., 2002), but has not been detected in vivo (Collins et al., 2001).

To determine whether in vivo co-expression of the M protein affects the transcription and replication of HRSV RNA analogues detected by metabolic labelling, we analysed the behaviour of an M protein variant (VM19), whose RNA-binding activity is reduced by 78 % compared with the normal M protein. We used the replication and transcription system developed by Hardy & Wertz (1998). The plasmids pWT5 and pM/SH produce HRSV viral RNA analogues, with 3' and 5' end HRSV A2 strain leader and trailer sequences, respectively, containing the promoters for virus replication and transcription. One (pM/SH) contains the intergenic region between the SH and M genes, which allows testing of the transcriptional anti-termination capacity of the M2-1 protein and thus whether the M protein exerts an effect on M2-1 protein activity.

Each plasmid was co-transfected in HEp-2 cells infected with vaccinia recombinant vTF-3 with plasmids under the control of the T7 RNA polymerase promoter expressing the cDNAs of L, P, N, M and M2-1 or M2-1 plus M2-2 proteins. The pL plasmid contained cDNA from the A2 strain L polymerase with the N1049D point mutation, which affects transcription termination (Cartee et al., 2003). The remaining plasmids contained Long strain sequences, except pN, which contained a point mutation present in A2 but not in the Long strain protein.

Results obtained with pM/SH (Fig. 6B) were similar to those observed with plasmid pWT5 (not shown) and were independent of the presence of the M2-2 protein. A slight effect was observed on virus replication and transcription with different M protein amounts (between 0·44 and 0·9 µg). These results concur with those obtained in a similar system (Collins et al., 2001).



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Fig. 6. Effect of M protein expression on HRSV RNA analogue transcription and replication. (A) Schematic representation of the DNA construct in the pM/SH plasmid, which expresses a dicistronic HRSV RNA analogue. Sizes and polarities are shown for the different RNAs generated during transcription and replication (Hardy & Wertz, 1998). (B) HEp-2 cells grown on an 8 cm2 plastic surface were transfection with 1·08 µg pM/SH and pGEM3N plasmids, 0·32 µg pL, 0·44 µg pGEM3P, 0·062 µg pGEM3.M2 or pGEM3M2-1 and 0·44, 0·6, 0·9 or 1·2 µg pGEM3M (M) or 0·6, 0·9 or 1·2 µg pGEM3M19 (M'). At 16 h after transfection, the RNA was labelled with 25 µCi [3H]uridine ml-1 in the presence of 10 µg actinomycin D ml-1. At 24 h post-transfection, cytoplasmic cell extracts were processed using a kit from Qiagen to purify the RNA. The RNAs were electrophoresed, on a 3·5 % acrylamide gel containing 7 M urea, dried and autoradiographed. (C) Total proteins were obtained from 10 % of the transfected cells after resuspension in SDS-PAGE buffer, boiling and centrifugation. The proteins were separated by 12 % SDS-PAGE and stained with Coomassie blue.

 
The size heterogeneity observed in the mRNAs is noteworthy (mainly in mRNA1 and the readthrough at the trailer sequence in mRNA2), according to the L polymerase properties used (N1049D). A more marked inhibitory effect on transcription and replication was found for the largest amount of M protein tested (1·2 µg). Overall, the M protein variant VM19 behaved like the normal M protein. Thus, the inhibitory effect detected at high protein concentration seems to be independent of the 78 % reduced RNA-binding capacity of the VM19 variant M protein, which had residues 156, 157 and 170 changed.

Capacity of the M protein to form oligomers
M protein expressed alone in HEp-2 cells could be cross-linked with glutaraldehyde (Asenjo & Villanueva, 2000) (Fig. 7). After incubation with the cross-linker, high molecular mass oligomers were assembled that were unable to enter a 10 % acrylamide gel. Formation of these homo-oligomers was independent of M protein RNA-binding properties, since the M protein variant VM23, whose RNA-binding capacity was only 8 % that of the normal M protein, was able to form these oligomers (composed of more than eight monomers, as determined by its retarded electrophoretic mobility). The VM25 M protein variant, which was unable to bind RNA (Fig. 4B), also formed these oligomers (not shown).



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Fig. 7. The RNA-binding capacity of HRSV M protein is not essential for its ability to form high-order oligomers. Proteins from soluble extracts of HEp-2 cells transiently expressing M protein or variant VM23 were cross-linked with 0·01 % glutaraldehyde (see Methods) and analysed at different times. The reaction was terminated by adding 0·2 M glycine. SDS-PAGE sample buffer was added and the proteins were separated by SDS-PAGE, then transferred to Immobilon membrane and analysed by Western blotting using a monospecific rabbit anti-M serum. Lane V, HRSV extracellular viral particles; lanes VM and VM23, the cross-linking kinetics of M protein and the VM23 variant, respectively. The electrophoretic mobilities of molecular mass markers M and VM23 proteins are indicated by single asterisks. The positions of the high-molecular-mass oligomers are indicated by two asterisks.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have shown that HRSV M protein, an abundant HRSV structural protein with an important role in viral particle formation, is an RNA-binding protein. This is the first description of this property for an M protein of a member of the Paramyxoviridae family and was shown experimentally in three assays that detected its ability to bind nucleic acids.

The HRSV Long strain M protein was shown to bind to both short (86 nt) and long (700–1200 nt) RNAs and was not sequence specific. This RNA binding was cooperative and saturable, with an apparent dissociation constant of 25 nM. The protein appeared to have two types of RNA-binding sites, which may be related to its capacity to bind RNA as a monomer, discrete oligomers (dimers, Fig. 1A) or high molecular mass oligomers (more than eight monomers, Fig. 7), although the two binding sites found could also be related to the presence of two M protein populations with different renaturation properties. The role of these M protein–RNA interactions during the virus growth cycle is unknown, but may be related to early host-cell transcription inhibition detected during HRSV infection (Ghildyal et al., 2003).

The M protein region containing the RNA contact residues was mapped by preparative UV cross-linking of the M protein with 32P-labelled riboprobes. After RNase digestion of the non-cross-linked probe, the protein was CNBr- and trypsin-treated, and the peptides separated and located on the M protein molecule after N terminus determination. As the exact fragment ends were unknown and could be determined only approximately, a broad region (84 of 256 total residues) between residues 90 and 173 was mapped as the M protein RNA-binding region, although additional sites in the last 50 C-terminal residues could not be ruled out.

To define this M protein region (90–173) more precisely, deletion variants were generated. Analysis of their RNA-binding capacities confirmed previous data. All M protein variants with C-terminal deletions showed RNA binding similar to that of the intact M protein except those with deletions among residues 209–216 and 186–256. Because the first deletion is contained in the second and by taking into account the trypsin maps (Fig. 2B, T2 peptide), a more C-terminal location should be expected for putative binding at the C terminus of the M protein. Thus, it seems that the decreased RNA-binding values found for these variants may indicate a requirement for the C-terminal end of the M protein for its RNA-binding capacity.

Variants with deletions between residues 121 and 130, 154 and 160, 156 and 166 and 162 and 171 showed decreased RNA-binding capacity. According to the rationale outlined above, this could be because only residues located between these positions are involved in binding. An assay of M protein substitution variants defined K and R at positions 121, 130, 156, 157 and 170 as the probable RNA contact residues, because in their absence but in the presence of other possible candidates (Y163, L164, R165 and residues 209–216 and/or 186–256) no binding was detected. Nevertheless, conformational changes could take place and, as consequence, these residues may not be able to bind RNA, a feature that could occur in other M protein variants.

The basic nature of residues 121, 130, 156, 157 and 170 indicates that the interaction with RNA is ionic, probably through RNA phosphate groups, as suggested by the lack of sequence specificity found for M protein binding. M protein from influenza virus also binds RNA through basic residues included in its nuclear location signal (Elster et al., 1997). Whether the basic residues determined here take part in a non-canonical nuclear localization signal in HRSV M protein remains an open question. For the M protein from Ebola virus, aromatic (F121), basic (R134) and hydrophobic (L132 and 158) residues have been found in contact with the ssRNA motif U-G-A (Gomis-Ruth et al., 2003).

All residues except K156 or 157 are conserved in all pneumovirus M proteins that infect humans, cows, sheep, mice and turkeys, including the recently described metapneumovirus (Van der Hoogen et al., 2002). This indicates that the RNA-binding domain is a structural characteristic of the M protein. Crystallographic studies are required to determine whether these residues contact RNA or are needed to maintain the RNA-binding domain structure.

The residues identified must combine to form the possible two binding sites found for the purified and renatured M protein. Conservation of protein structure around the RNA-binding motif suggests an essential role for this interaction in M protein function during the virus infection cycle. HRSV M protein is reported to be present in the nucleus of infected cells at early but not late times post-infection. This is related to the inhibitory transcription capacity of the HRSV-infected nuclear extract (Ghildyal et al., 2003). The RNA-binding capacity described here for the HRSV M protein may concur with these results, although the relevance of the transcriptional cellular inhibition for viral metabolism is not known.

Although HRSV infection does not ‘shut off’ cell macromolecule synthesis, global alteration has been described in host epithelial cell transcription of at least 3300 cellular genes as a consequence of HRSV infection (Zhang et al., 2003). HRSV M protein should be able to form pore-like structures according to its capacity to form high-molecular-mass oligomers in the absence of its RNA-binding capacity. It is possible that part or all of the residues involved in the M protein RNA-binding capacity are required for its nuclear cell location and/or for interfering with cellular RNA metabolism once located in the nucleus.

In other viral systems, two distinct functions are described for M protein–RNA interactions. In the murine coronavirus, the M protein interacts with RNA containing a short cis-acting RNA element, the packaging signal, in the absence of the N protein (Narayanan et al., 2003). In rabies virus, the M protein regulates the balance between transcription and replication by inhibiting the former and activating the latter, although it is not known whether the M protein has RNA-binding capacity (Finke et al., 2003). The rabies virus M protein activity is independent of its compacting RNP capacity, which renders RNP containing vRNA or cRNA inactive for RNA synthesis. This property is also shown by HRSV M protein in vitro (Ghildyal et al., 2002), but not in the HRSV-based RNA analogue system, unless large amounts of M protein are expressed (Collins et al., 2001; this paper).

Although HRSV RNA analogue transcription should be dependent on its replication capacity, this is difficult to analyse precisely if M protein co-expression affects viral transcription and replication of HRSV RNA analogues, as this may be sufficient to affect the viral growth cycle once amplified during infection. To analyse this possibility, HRSV RNA analogues suitable for virus transcription- or replication-independent studies must be constructed and assayed. An essential role in virus replication has none the less been shown for one of the regions that forms the influenza virus M protein RNA-binding domain (Liu & Ye, 2002).

The transcription and replication inhibition mediated by large amounts of M protein resembles that seen in HRSV-infected HEp-2 cells, in which M protein binding to the RNPs and its corresponding transcription inhibition are observed late in infection (Barik, 1992; Ghildyal et al., 2002) when M protein levels in HRSV-infected cells are higher. In accordance, inhibition of HRSV RNA analogues was also found when 1·2 µg of the pM plasmid was co-transfected in the replication and transcription assay. This effect appears similar to that of RNP compaction observed for rabies virus M protein. Since this effect was also produced by an RNA-binding-impaired M protein variant (VM19, which displayed 22 % of the M protein RNA-binding capacity), it seems that M protein-mediated nucleocapsid compaction is independent of its RNA-binding capacity, in which residues 156, 157 and 170 are important.

Further experiments are needed to determine whether HRSV M protein acts similarly to mouse coronavirus M protein (Narayanan et al., 2003). It must also be determined whether HRSV M protein shows specificity for the trailer sequences and whether it can form VLPs that include RNAs containing such sequences. This activity may involve M protein residue combinations distinct from those required for cell transcription inhibition.

In conclusion, M protein RNA-binding capacity, mediated by the residues described here, may be a good target for specific HRSV antiviral compounds, if the interaction described here plays an important function in the virus growth cycle.


   ACKNOWLEDGEMENTS
 
We thank R. Martinez and M. I. García-Albert for excellent technical assistance, Drs J. Ávila and G. Wertz for critical reading of the manuscript and C. Mark for language editing. We are also indebted to Dr Wertz for kindly providing pWT5, pM/SH and pL plasmids. L. R., I. C. and A. A. received fellowships from the Instituto de Salud Carlos III of the Spanish Ministry of Health. This work was supported by the Instituto de Salud Carlos III intramural project MPY00/204 to N. V.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 8 October 2003; accepted 6 November 2003.