INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus of the Paramyxoviridae family, is the most common infectious agent in bronchiolitis and pneumonia requiring hospitalization of infants and young children. Immunocompromised and elderly people are also severely affected. No vaccine or specific antiviral treatment is available at present (11). The best way to control these infections, according to the characteristics of the human populations mainly affected, is the use of different agents as live attenuated vaccines (38), humanized neutralizing monoclonal antibodies (21), and antiviral compounds (27).

The HRSV nucleocapsids are, as in all paramyxoviruses, structural components and functional units for replication and transcription processes (11). The nucleocapsid proteins are thus multifunctional, serving as ideal targets for the action of antiviral compounds and for conveying attenuation mutations. The design of both types of reagents would be aided by understanding nucleocapsid protein functions.

HRSV nucleocapsids are composed of the negative-sense RNA genome (15,222 nucleotides long), tightly bound to the N protein, the large polymerase protein (L), the phosphoprotein (P), and probably the M2-1 protein (11, 16). The RNA polymerizing activity of the viral nucleocapsids is due to L protein, for which activity P protein is essential (41). N protein plays a histone-like function, since synthesis of viral RNA always occurs on ribonucleoprotein templates. P protein also acts as a chaperone of N protein, maintaining it competent for encapsidation (30). M2-1 protein enhances the processivity of the viral polymerase (9, 10) and its readthrough of intergenic junctions (17). These properties identified M2-1 protein as an elongation and antiterminator transcription factor, a constituent of the nucleocapsids. The protein is encoded by the M2 gene, present only in the pneumoviruses, which codes for an mRNA containing two open reading frames (ORFs). ORF1 encodes the 22K structural protein, also known as M2-ORF1 or M2-1 protein (194 amino acids). ORF2 encodes M2-2 protein (90 amino acids), detected in HRSV-infected cells, which has a regulatory function in the balance between viral transcription and replication (1, 6, 8).

The M2-1 protein was long ago described as a structural membrane phosphoprotein that could be present as different species in HRSV-infected cells (20, 23). These species could differ in the number of intramolecular disulfide bonds formed among its four cysteine residues (23). Three of the four cysteine residues are at the amino-terminal end, forming a C₃H₁ motif that is highly conserved among members of the genus. In other proteins, this motif has been shown to bind Zn ions (40). Mutation at the predicted Zn coordinating residues of the C₃H₁ motif (C7, C15, and H25) abolished M2-1 enhancement of transcriptional readthrough (18). Interaction of M2-1 with the N protein has been also described in coexpression experiments (16).

Here we present evidence that M2-1 is an RNA binding protein, able to bind the RNA leader specifically, whose binding capacity is modulated by phosphorylation. The amino acid residues in contact with the RNA and those modified by phosphorylation have been located.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells and viruses. HEp-2 cells, COS1 cells (CV1 cells containing the simian virus 40 T antigen), HRSV Long strain, and the vaccinia virus recombinant vTF-3 were used throughout this study in experimental conditions previously described (35, 37).

Transfection. cDNAs of different viral proteins, all cloned in pGEM3, were transiently expressed in the vaccinia virus-based expression system under the control of the T7 RNA polymerase promoter (vTF-3). The corresponding soluble proteins were prepared as described elsewhere (37). Briefly, cell cultures were recovered by low-speed centrifugation, twice washed with phosphate-buffered, and then homogenized in 10 mM Tris-HCl (pH 7.5)-140 mM NaCl-5 mM EDTA-1% Triton X-100 and-1% deoxycholate. The supernatant fraction obtained after centrifugation in a minicentrifuge (15 min, 4°C) was considered the soluble protein fraction. For some variants, insoluble proteins were obtained after removal of soluble proteins; they were further solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.

Plasmids. The cDNAs corresponding to N, P, and M2 genes of HRSV Long strain, cloned in the corresponding pBSV9 recombinant plasmids (24), were subcloned as HpaI-StuI fragments in the SmaI site of the plasmid vector pGEM3.

M2-1 protein purification. HEp-2 cells were infected with HRSV, and the microfilament-containing protein fraction and extracellular viral particles were obtained as previously described (32). The proteins contained in the different fractions were separated and characterized in SDS-gels (12% acrylamide) containing 0.4% Coomassie blue in the electrophoresis buffer or after negative staining (14). The piece of gel containing the M2-1 protein was excised, and the protein was electroeluted. The protein was then precipitated with acetone and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 7.5]) containing 6 M guanidine chloride. The protein was renatured by chromatography through an acrylamide column using TE as the running buffer. The fractions containing the M2-1 protein were detected after SDS-PAGE, pooled, and densitometrically quantified by comparison to known amounts of bovine serum albumin (BSA), separated in the same gel.

Preparation of labeled riboprobes. The ³²P-labeled riboprobes used were prepared by in vitro transcription of the corresponding pGEM3 recombinant plasmids, using T7 or SP6 RNA polymerase and adding [-³²P]CTP to the reaction (25). In this way, ³²P-labeled plus- or minus-polarity RNAs were obtained, corresponding to genes P (1,000 nucleotides) and N (1,200 nucleotides). A plus-polarity RNA (700 nucleotides) containing the leader sequence plus three extra G's at the 5' end was obtained using plasmid pL digested with NcoI in the in vitro transcription reaction.

UV cross-linking assay. M2-1 protein (10 to 180 ng) was mixed with 1 to 5 ng of the indicated riboprobe (specific activity, 30,000 Cerenkov cpm/ng) and incubated in binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 100 µM ZnCl₂, 4 mM dithiothreitol). The RNA-protein complex was UV cross-linked as previously described (3). After treatment with bovine pancreas RNase A (60 µg/ml), SDS-PAGE (12% acrylamide gel) was carried out. The gels were stained with Coomassie blue and autoradiographed.

Gel retardation assay. The protein and riboprobes were incubated as indicated above and then electrophoresed in a gel containing 3.5% acrylamide and 0.5× Tris-borate-EDTA buffer at 100 V. The gels were fixed with 95% ethanol, dried, and autoradiographed. The amount of radioactivity present in the riboprobe position was quantified by a PhosphorImager. This procedure was used for kinetic studies to measure the formation of the protein-RNA complex. The protein concentration at which 50% of the labeled RNA formed an RNA-protein complex was defined as the apparent K_d (dissociation constant).

Northern-Western assay. Protein (30 to 40 µg) obtained after transfection experiments was separated by SDS-PAGE and electrotransferred to nitrocellulose paper. The paper was washed with 100 mM NaCl-40 mM Tris-HCl (pH 8.0), and the proteins were renatured by incubation in the same buffer containing 0.02% PVP-40 (polyvinylpyrrolidone), 0.02% BSA, 0.02% Ficoll 400, 0.1% Triton X-100, and 50 µM ZnSO₄ for 3 h at 20°C. The ³²P-labeled riboprobe was added in the same buffer without ZnSO₄ and containing yeast RNA (1 µg/ml). After 2 h of incubation, the probe was removed and the paper was washed several times with incubation buffer. The dried paper was exposed in a PhosphorImager.

Chemical and enzymatic treatments of the M2-1 protein. The cross-linking reaction described above was carried out with 80 ng of purified M2-1 protein and 5 ng of ³²P-labeled riboprobe. After RNase digestion and precipitation with acetone, the labeled M2-1 protein with associated radioactivity was mixed with 100 to 300 ng of electroeluted M2-1 protein. The mixture was then treated with 14 or 28 mg of cyanogen bromide (CNBr) per ml in 50% formic acid at 37°C overnight or for 4 h (15). After incubation, the sample was vacuum dried, neutralized by several cycles of resuspension in 0.1 M NH₄HCO₃ (pH 8.3), and again vacuum dried. When the sample was at neutral pH, it was resuspended in 50 µl of electrophoresis sample buffer. In other cases, the protein was treated with N-bromosuccinimide or with V8 protease as previously described (15, 39).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

M2-1 is an RNA binding protein. To determine whether HRSV nucleocapsid proteins are able to interact with viral RNA, protein fractions from HRSV-infected HEp-2 cells were chromatographed through Sepharose 4B containing covalently bound monoclonal antibodies specific for the different viral proteins. The viral proteins purified in this way were unable to bind ³²P-labeled RNAs by cross-linking and by retardation in gel electrophoresis assays. These results could be due to the denaturation of the purified proteins, to the fact that RNA is already bound to them, or to both possibilities. To avoid this difficulty, we first purified the nucleocapsid proteins by SDS-PAGE and then renatured them. As a nucleocapsid source, a microfilament fraction and extracellular viral particles from infected HEp-2 cells were used, because nucleocapsid proteins are in a higher proportion in these fractions than in other subcellular fractions. The protein composition of the protein fractions obtained from HRSV-infected HEp-2 cells and the purity of the viral nucleocapsid proteins after purification and renaturation are shown in Fig. 1A and B. 

View larger version (93K): [in this window] [in a new window]   FIG. 1.   Purified and renatured M2-1 protein binds to RNA, as shown by cross-linking with UV and in electrophoresis gel retardation experiments. (A) SDS-polyacrylamide gel stained by Coomassie blue showing the protein composition of 20 µg of protein contained in extracellular viral particles (V) and soluble (S), microfilament-bound (M), and insoluble (I) protein fractions from HRSV-infected HEp-2 cells. (B) Purity of viral proteins after renaturation and quantification. The BSA slots correspond to 92, 231, 462, 693, and 926 ng of pure BSA. The following slots correspond to different volumes (2 and 4 µl) of purified and renatured N, P, M, and M2-1 proteins. The calculated protein concentrations varied between 0.1 and 0.2 mg/ml. The electrophoretic mobility of HRSV proteins and of molecular weight markers (MW; expressed in kilodaltons) are indicated at the left and right, respectively. (C and D) The indicated amounts (nanograms) of purified M2-1 protein were incubated with 2 ng of RNA (1,000 nucleotides long) labeled with 32P (35,000 Cerenkov cpm/ng) under the indicated conditions. Samples were then UV cross-linked and analyzed by SDS-PAGE following RNase digestion (C) or run in a 5% acrylamide gel (D). Asterisks indicate the electrophoretic mobility of M2-1 and its dimers.

M2-1 protein specifically binds to short RNAs containing the 5'-end leader sequence. Different amounts of the renatured M2-1 protein were assayed by mobility retardation in gel electrophoresis, using 1 ng of ³²P-labeled riboprobes corresponding to plus (mRNA) and to minus (viral RNA) polarities of HRSV P and N genes (1,000 and 1,300 nucleotides, respectively) and to plus-polarity RNA containing the HRSV leader plus three extra G's at the 5' end (700 nucleotides) and therefore with the structure at the 5' end of the viral cRNA. Similar kinetics were obtained in all cases tested (Fig. 2A). The protein is also able to bind to other long RNAs unrelated to HRSV (data not shown); M2-1 protein therefore binds without sequence specificity to long RNAs. The apparent K_d, calculated from quantification of the results, is 30 nM for these long RNAs. The retardation of the probes in all cases is due to binding of the protein to a long RNA since binding can be competed for by different unlabeled long RNAs but not by short yeast RNA, which has an average size of 100 nucleotides (data not shown).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   M2-1 protein binds specifically to HRSV leader RNA. Different quantities of purified M2-1 protein were incubated with 1 ng of 32P-labeled (30,000 Cerenkov cpm/ng) long riboprobes (vRNA [viral RNA] and mRNA) corresponding to the RNAs for the P gene in minus and plus polarities, respectively (cRNA corresponds to 700-nucleotide positive-polarity long RNA containing the leader sequence at its 5' end) (A) or with 0.1 ng of 32P-labeled (100,000 Cerenkov cpm) short RNAs (leRNA, 90 nucleotides long, containing the leader sequence at its 5' end; sRNA, a pGEM4-derived RNA, 50 nucleotides long) (B) with increasing amounts of M2-1 protein. Lanes labeled 0, 1, 2, 3, 4, and 5 correspond to the absence of protein or to the addition of M2-1 protein at final concentrations of 18, 36, 54, 109, and 218 (A) and 18, 36, 72, 145, and 290 (B) nM. The amount of radioactivity was quantified by PhosphorImager. The results represent the mean of several experiments with each riboprobe.

Mapping of the M2-1 RNA binding domain. After preparative cross-linking and RNase digestion, the labeled protein was treated with CNBr, N-bromosuccinimide, or V8 protease. The peptides generated after the different treatments were separated by electrophoresis and visualized by staining with amido black; those labeled with ³²P were detected by autoradiography. Figure 3A shows the result obtained after treatment with CNBr and V8 protease digestion. The amino-terminal sequence of the peptides generated was determined. The data from several experiments (summarized in Fig. 3B) indicate the calculated sizes for the fragments and the amino acid residues located at their NH₂-terminal ends, although the calculated fragment sizes may be overestimated due to their smallness and to the possible presence of oligoribonucleotides bound to them. The approximate locations of these fragments on the M2-1 molecule are shown, although their C-terminal ends are unknown (Fig. 3C). These results indicate that at least some of the residues in contact with the RNA are between positions 55 and 75 but do not rule out the existence of others around this region. The RNA binding region was thus considered to be located between amino acid residues 40 and 80. It also appears that the zinc finger domain does not contain the residues in direct contact with the RNA since the V3 fragment, which starts at the residue at position 10, contains no associated radioactivity.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Location of M2-1 protein amino acids residues in contact with RNA. (A) Preparative RNA binding assays were carried out with 150 ng of M2-1 protein and 8 ng of 32P-labeled RNA (1,000 nucleotides long). After UV cross-linking, RNase A digestion, and acetone precipitation, the covalently bound RNA-M2-1 protein was resuspended in TE buffer containing 300 ng of purified M2-1 protein. The mixture was treated with 28 mg of CNBr per ml in 50% formic acid [CNBr(1)] or with 1 µg of protease V8 (V8) in 50 mM NH4HCO3 for 4 or 2 h, respectively, at 37°C. The peptides generated were separated in SDS-polyacrylamide gels containing 6% urea and Tricine in the electrophoresis buffer (31). After electrophoresis, peptides were transferred to Inmobilon membranes and detected by amido black staining (S) or by PhosphorImager (32P). (B) NH2-terminal sequences for the peptides. Sizes of molecular weight (MW) markers are indicated in kilodaltons. (C) Representation of the M2-1 protein molecule and the mapping of unphosphorylated and phosphorylated peptides to it. The dashed lines at the C-terminal ends of fragments indicate that their exact end positions are unknown. The amino acid sequence in the M2-1 protein RNA binding region is indicated. The residues starting the different phosphorylated peptides are also indicated in that sequence.

Deletion variants of M2-1 protein and their RNA binding capacities. To confirm that between amino acids 40 and 80 of M2-1 protein are those that contact directly RNA and to pinpoint the residues essential for RNA binding, we tested the RNA binding capacity of M2-1 protein and different deletion variants by Northern-Western blotting using M2-1 proteins transiently expressed in the vaccinia virus-based expression system. The cDNA corresponding to the HRSV M2 gene was expressed in this system, and a protein with a mobility slower than that of M2-1 present in the purified virions was observed. This protein has been identified as phosphorylated M2-1 protein (see below).

View larger version (45K): [in this window] [in a new window]   FIG. 4.   M2-1 protein deletion variants and their RNA binding capacities determined by Northern-Western assays. (A) Diagram representation of M2-1 protein indicating the amino acid residues deleted in the different variants as a broken lines. The residues unrelated to M2-1 protein and added as a consequence of the cloning procedures are also shown. Right and left parts of panels B to D correspond to Northern-Western and Western assays, respectively, of 50 µg of total protein. The different M2-1 protein variants are indicated by asterisks in both assays. In panel B, lane V corresponds to extracellular viral particles, and lanes 1, 2, 3, 4, 5, and 6 correspond to soluble proteins from transfections with pGEM3 and pGEM3 recombinants M2-1, del5, del10, del16, and del26, respectively. In panel C, lanes, 2, 3, 7, 8, 9, and 10 correspond to insoluble proteins from transfection with pGEM3 recombinant plasmid M2-1, del5, EcoRV, EcoRVts, EcoRV-NdeI, and DpnI constructs. In panel D, lane 11 corresponds to soluble protein from transfection with the pGEM3 recombinant plasmid PstI. The center portion corresponds to a longer exposure of the Northern-Western blot; the asterisk indicates the PstI variant M2-1 protein position. The Western blots were developed with monospecific anti-M2-1 protein serum except for the insets containing lanes 9 and 3 in panel C, which were developed with anti-HRSV serum.

M2-1 protein is phosphorylated mainly when expressed in the absence of other viral proteins. HEp-2 cells infected with the vaccinia virus recombinant vTF-3 were transfected with plasmid pGEM3 or pGEM3-M2. After labeling with [³⁵S]methionine, cell extracts were prepared, and the proteins were analyzed by SDS-PAGE. In pGEM3-M2-transfected cells, we detected a protein that is absent in the cells transfected with pGEM3. This protein showed a mobility slower than that of M2-1 protein present in purified virions (Fig. 5A, lane 2, ³⁵S). Previous results (23) described a 24-kDa structural phosphoprotein, in addition to P protein, which may be M2-1 protein. The change in electrophoretic mobility observed for the transiently expressed M2-1 protein may thus be due to phosphorylation. To test this, the above experiment was repeated but with [³²P]orthophosphate labeling. ³²P labeling was found in the protein expressed by the recombinant pGEM3-M2 plasmid (Fig. 5A, lane 2, ³²P).

View larger version (63K): [in this window] [in a new window]   FIG. 5.   M2-1 protein is phosphorylated at serine and threonine residues when is expressed in transfected and HRSV-infected HEp-2 cells. Direct or indirect interactions with P protein modulate M2-1 protein phosphorylation. (A) HEp-2 cells were transfected (T) with plasmid pGEM3 (lane 1) or pGem3-M2 (lane 2), mock (U) or HRSV (I) infected, and labeled with [32P]orthophosphate. In the transfection experiment, the protein was also labeled with [35S]methionine. Soluble (S) and microfilament-associated (M) protein fractions were obtained. The proteins were separated by SDS-PAGE and visualized in a PhosphorImager. The viral proteins in extracellular viral particles (V) stained by Coomassie blue (C) are shown at the right. (B) (Left) The piece of gel containing the phosphorylated protein, with electrophoretic mobility slower than that of M2-1 protein of extracellular viral particles expressed by the recombinant plasmid in the transfection experiment (T), was excised from the gel and hydrolyzed with 6 N HCl for 2 h at 110°C. The resulting amino acid residues were separated by two-dimensional electrophoresis in thin layer and characterized by their 32P labels. Unlabeled phosphothreonine (P-T), phosphoserine (P-S), and phosphotyrosine (P-Y) were included as markers and detected with ninhydrin. (Right) Phosphorylated protein expressed by pGEM2-M2 (T), and polypeptides with the same electrophoretic mobility in the microfilament fraction of infected cells (I) and in virions (V), after excision from the gel and CNBr treatment. After renaturation, the peptides were separated in SDS-20% urea acrylamide gels, and those in phosphorylated form were detected by a PhosphorImager. Sizes are indicated in kilodaltons. (C) HEp-2 cells were infected with recombinant vaccinia virus vTF-3 and transfected with pGEM3 (lane 1), with pGEM3-M2 (lanes 2 to 5), and with pGEM3-M2 plus pGEM3-N (lane 3), pGEM3-N and pGEM3-P (lane 4), and pGEM-P (lane 5). The cultures were [35S]methionine labeled, and the soluble proteins were analyzed by SDS-PAGE before (left) or after (right) immunoprecipitation with rabbit anti-HRSV serum.

Deletions variants of M2-1 protein and their phosphorylation capacity. When M2-1 protein and its variants were transiently expressed, differences in solubility and in the presence of two molecular species were observed (Fig. 6A). To determine whether the presence of a second protein species is due to phosphorylation, transfected cells were labeled with [³²P]orthophosphate (Fig. 6B), their specific activities were determined, and the results obtained were compared to those for the normal M2-1 protein (Fig. 6; Table 1).

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Phosphorylation of M2-1 deletion variant proteins. HEp2-cells were infected with vaccinia virus recombinant vTF-3 and transfected with the following or with pGEM3 recombinant plasmids: M2 (lane 1), del5 (lane 2), del10 (lane 3), del16 (lane 4), del26 (lane 5), EcoRV (lane 6), EcoRV-NdeI (lane 7), EcoRVts (lane 8), and DpnI (lane 9). The cultures were labeled with [32P]orthophosphate, and soluble (S) and insoluble (I) protein fractions were isolated, analyzed in SDS-15% polyacrylamide gels, and visualized by Coomassie blue staining (A, left) or by autoradiography (A, right), [35S]methionine-labeled cultures; (B, right and left; [32P]orthophosphate-labeled cultures). M, size markers; V, viral proteins contained in extracellular viral particles. Table 1 shows specific activity (phosphorylations), relative to that of control M2-1, for protein variants.

Identification and localization of phosphorylated residues on the M2-1 molecule. M2-1 protein transiently expressed in HEp-2 cells with the vaccinia virus-based system was labeled with [³²P]orthophosphate or [³⁵S]methionine, fractionated by gel electrophoresis, transferred to Inmobilon membranes, and excised. Proteins were then treated with CNBr and N-bromosuccinimide. Labeled peptides were separated and further identified by autoradiography. In accordance with the cleavage specificity of the chemical agents used, peptide size, and methionine residue distribution in the M2-1 molecule, the phosphorylated residues were tentatively located at the NH₂-terminal region between amino acid residues 40 and 100 (data not shown). Concurring with this location, the deletion variants lacking amino acid residues between positions 85 and 166, 110 and 166, and 110 and 194 were phosphorylated at a higher level than the normal protein. The slight phosphorylation for del10 and the lack of phosphorylation found for del16 and del26 indicated that the phosphorylated residues could be located between residues 48 and 85, taking into account that there are no serine and threonine residues between amino acid residues 32 and 48 of M2-1 protein. Previous experiments indicated (36) that purified HRSV particles contain casein kinase II (CKII)-like activity, able to phosphorylate their P protein among others proteins present in the purified viral particles. One of these proteins has the same electrophoretic mobility as the phosphorylated M2-1 protein. The phosphorylated amino acids of M2-1 may thus be modified by CKII protein.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

M2-1 protein has been described as a putative nucleocapsid structural component that acts as a transcription factor by increasing the processivity and readthrough of intergenic sequences by the viral polymerase constituted by the L and P proteins (9, 10, 17). Nevertheless, the increased readthrough of the viral polymerase in the presence of the M2-1 protein does not increase the replication products of HRSV RNA analogs (13). M2-1 incorporation into the viral polymerase complex composed of L and P proteins is thus not related to a switch of polymerase activity from the transcription to the replication mode (10).

In its NH₂-terminal sequence, the protein has a Zn finger formed by three cysteine residues and one histidine residue, like several other transcription factors (40). Like them, M2-1 has RNA binding capacity, shown here using three different assays.

By testing long (700- to 1,300-nucleotide), RNAs we found that M2-1 protein may bind to different RNAs with the same affinity, suggesting a histone-like activity for this protein similar to that ascribed to the N protein in paramyxoviruses (12). M2-1 shows also specificity for certain RNA sequences. To determine sequence specificity, we tested shorter RNA molecules and found M2-1 to specifically recognize the leader RNA (44 nucleotides) at the 5' end of short RNAs (80 nucleotides). The distinct affinities of the M2-1 protein for long and leader RNAs could be due to the binding of different M2-1 oligomers (monomers and trimers or dimers and hexamers) for each type of RNA. It may also be due to the increased number of nonspecific binding sites in the long RNAs. On the other hand, the phosphorylation state of M2-1 may change the affinity and perhaps the specificity of the protein for the different RNAs. The M2-1 RNA binding domain has been mapped outside the zinc finger by preparative cross-linking of the protein to labeled riboprobes. In addition, an M2-1 protein variant (del5) without the Zn finger (located in the first 30 amino acid residues) binds to RNA with greater affinity than does the intact M2-1 protein, according to their expression levels of both proteins (Fig. 4C), indicating the the Zn finger is not essential for M2-1 RNA binding. The amino acid residues in contact with the RNA are located between residues 59 and 85, as indicated by binding capacities of variants lacking residues 32 to 58. In addition, residues at positions 59 to 62 may be sufficient for RNA binding, as indicated by positive RNA binding by the PstI deletion mutant. Nevertheless, the RNA binding capacity of the residues added in the cloning procedure involved in obtaining variant PstI cannot be excluded. Other residues in contact with the RNA, between residues 62 and 85, cannot be ruled out. In addition, the binding capacity of the protein variants devoid of residues 85 to 194 suggests that this part of the molecule is not important for M2-1 binding to RNA. The affinities of the different variants remain to be determined to ensure the importance of different residues in RNA binding.

In contrast, the retroviral nucleoprotein interacts with viral RNA through a Zn finger. In human immunodeficiency virus, basic residues in the first and second Zn fingers (33) are involved in electrostatic contacts with RNA and may be responsible for the specificity of RNA encapsidation. Other Zn fingers are not involved in the binding to nucleic acids; replication protein A (26), for example, binds to single-stranded DNA through molecular regions located outside the Zn finger. These metal binding domains have been identified in several proteins of different origins (5); although they are able to bind nucleic acids in some cases, they also facilitate interactions between proteins and other macromolecules. The metal binding domain can thus also be involved in interactions between protein monomers (28). One can therefore speculate that the metal binding domain of M2-1 plays a role in the dimer formation detected in the UV cross-linking experiments. Its presence could also stabilize the native protein conformation. It appears that the native conformation of the RNA binding domain is facilitated by the presence of Zn ions during the renaturation process, in accordance with the importance of this domain in global structure of the protein. Thus, substitution of residues C7, C15, and H25 predicted at those that coordinate Zn and prevent M2-1 readthrough transcription activity, phosphorylation, and interaction with N protein indicates that the C₃H₁ motif is essential for maintaining the functional integrity of the protein (18).

In the M2-1 proteins of human, bovine, and ovine virus origin, amino acid conservation between residues 59 and 80 is 95% (2). When the comparison includes the mouse pneumovirus M2-1 protein (1), there is only 45% similarity between residues 59 and 70 (residues S58, E/D59, I/V60, S61, G62, R68, and T69). Between residues 70 and 80, the conservation is 90% among all strains. The majority of these residues have an aromatic and hydrophobic nature (Fig. 7), similar to those in contact with the RNA in other RNA binding domains (5). The secondary structure of these binding region is a  sheet, according to Chou and Fasman predictions (7). For the amino acid residues at positions 50 to 62, an -helix structure is predicted (7). These secondary structures have been found in the RNA binding domains of other RNA binding proteins (5).

View larger version (17K): [in this window] [in a new window]   FIG. 7.   Comparison of pneumovirus M2-1 protein primary structures, showing conservation of the RNA binding domain and phosphorylatable residues. (A) Amino acid sequences of pneumovirus M2-1 proteins of human (A and B subgroup), ovine, bovine, and murine origins (1, 2). The RNA binding domain is boxed; phosphorylated residues are underlined; the last residue from each sequence is indicated in boldface. (B) Secondary structure prediction of M2-1 protein by the Chou and Fasman method (7). The rectangles represent blowups of the M2-1 protein NH2-terminal region showing the effect of substitutions T56E and S58E on its secondary structure.

It is noteworthy that the phosphorylatable residues, whose modification changes the protein RNA binding capacity, are also located in the M2-1 protein RNA binding domain. M2-1 appears to be phosphorylated at T56 and S58, with modification at these residues responsible for 96 to 98% of M2-1 protein phosphorylation.

T56 has the recognition consensus sequence described for the cellular protein kinase CKII (29), and its modification allows the same protein kinase to phosphorylate S53. T56 is not conserved in the mouse pneumovirus M2-1; in this case there is a threonine at position 52, with a consensus recognition sequence for CKII. In this protein, no additional residues could be modified after phosphorylation at T52. The amount of phosphorylated serine is higher than that of phosphorylated threonine; thus, additional serines seem to be modified. We therefore prepared the mutant T56A, S58A, which lacks 96 to 98% of the corresponding phosphorylation found in the EcoRV variant. This indicates that simultaneous phosphorylation at T56 and S58 is responsible for 96 to 98% of M2-1 phosphorylation. Other possibilities nonetheless remain open, as phosphorylation at these residues may be essential for further phosphorylation at a distant site located between amino acid residues 40 and 100, or replacement of these phosphorylatable residues by alanines, like the presence of del10 and del16, may disrupt the protein conformation essential for phosphorylation.

It is nevertheless clear that after removal of these residues, other threonine or serine residues are phosphorylated, since 2 to 4% of the phosphorylation level of the EcoRV mutant was observed. In the M2-1 protein sequence, S and T residues contained in the EcoRV variant with a signal recognition sequence for known kinases are S2 (a residue present in a sequence similar to that of the serine phosphorylated in the nucleoprotein of influenza virus [4]), S108 (CKI), and T180 (CKII). It is likely that this threonine residue or the serines at positions 2 and 108 are also modified, although further experiments are needed to probe this point.

M2-1 protein phosphorylation decreases its RNA binding capacity at least 5- to 10-fold in Northern-Western assays using long RNAs (data not shown). According to this result, modification in the predicted secondary structure of the M2-1 protein may occur when T56 and S58 are replaced by glutamic acid (Fig. 7).

M2-1 protein increases the processitivity of the viral polymerase intragenic (9, 10) and its readthrough capacity on all gene junctions, except at the junction leader-NS1 gene (13). It is tempting to speculate that the M2-1 protein capacity to bind leader RNA, present in short RNAs, should be related to the RNA polymerase ability to start RNA synthesis at the boundary leader-NS1 gene during viral transcription. In this point, it would be important to test the ability of M2-1 variants, mainly new ones with deletions in the RNA binding domain, for transcription processivity and readthrough. These and other intriguing possibilities are now being followed up. Because the direct or mediated interaction of M2-1 with P protein may determine the M2-1 phosphorylation level, the formation of these complexes could also modulate M2-1 protein RNA binding capacity.