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
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ABSTRACT |
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INTRODUCTION |
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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 NM 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.
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METHODS |
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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 HpaIStuI 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 712714 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 (10180 ng) were mixed with 15 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 (3040 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.
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RESULTS |
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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 120140, 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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Substitution variants were then constructed (Fig. 4A). Residues between 120 and 172 were replaced, concentrating on positions 154167, 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 186256, 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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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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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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DISCUSSION |
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The HRSV Long strain M protein was shown to bind to both short (86 nt) and long (7001200 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 proteinRNA 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 (90173) 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 209216 and 186256. 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 209216 and/or 186256) 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 proteinRNA 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.
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ACKNOWLEDGEMENTS |
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Received 8 October 2003;
accepted 6 November 2003.