1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
2 MRC Virology Unit, Institute for Virology, Church Street, Glasgow G11 5JR, UK
3 The Centre for Infectious Diseases, Wolfson Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, UK
Correspondence
José A. Melero
jmelero{at}isciii.es
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ABSTRACT |
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MAIN TEXT |
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Four viral proteins are essential for transcription of the HRSV genome: (i) the nucleoprotein (N), which associates with the viral and antigenomic RNA to form the ribonucleoprotein complex; (ii) the viral RNA-dependent RNA polymerase (L); (iii) the phosphoprotein (P), a cofactor of the viral polymerase; and (iv) the 22 kDa (22K) or M2-1 protein. Only the first three proteins, N, P and L, are required for replication of the genome.
The 22K protein is encoded by the first open reading frame (ORF1) of the M2 gene, which is unique to viruses of the subfamily Pneumovirinae. It was originally proposed as a second matrix protein based on solubility properties (Huang et al., 1985) and thus it was renamed M2-1. However, the 22K protein associates with characteristic cytoplasmic inclusions found in HRSV-infected cells that also contain at least N and P as other viral components (García et al., 1993
). In agreement with this subcellular localization, it has been reported that the 22K protein acts as a transcription elongation factor, allowing the synthesis of full-length mRNAs (Collins et al., 1996
). In addition, the 22K protein enhances read-through transcription at gene junctions to generate polycistronic RNAs (Hardy & Wertz, 1998
). It is possible that both effects reflect a transcription antitermination activity of the 22K protein (Fearns & Collins, 1999
) that functions by a presently unknown mechanism, although it has been reported that the 22K protein interacts with the P protein (Mason et al., 2003
) and that it binds RNA (Cuesta et al., 2000
) without sequence specificity (Cartee & Wertz, 2001
).
The 22K protein exists in at least two different isoforms identified by one-dimensional SDS-PAGE. The slower-migrating 24 kDa form is phosphorylated at Ser-58 and Ser-61 and is the most abundant isoform when expressed in the absence of other HRSV components (Cartee & Wertz, 2001). In contrast, the faster-migrating 22 kDa isoform is not phosphorylated and is the most abundantly expressed species in HRSV-infected cells.
The 22K sequence has a Cys3-His1 motif (C-X7-C-X5-C-X3-H) spanning aa 725 that has been proposed to bind zinc (Hardy & Wertz, 2000), by analogy to a similar motif found in the transcription factor Nup475 (Worthington et al., 1996
). Critical residues in this motif are essential for protein function in a minireplicon system (Hardy & Wertz, 2000
; Zhou et al., 2003
) and for virus infectivity (Tang et al., 2001
).
To gain structural information about the 22K protein, two sets of specific monoclonal antibodies (mAbs) were prepared: (i) mAbs 22K1 to 22K7 (Fig. 1a) were obtained from BALB/c mice immunized with affinity-purified 22K protein expressed in HRSV-infected HEp-2 cells; and (ii) mAbs 65, 112, 129 and 312 (Fig. 1a
) were obtained from BALB/c mice inoculated with a GST22K chimera expressed in bacteria.
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To map the antibody epitopes in the 22K primary structure, a set of 22K deletion mutants was prepared. The ORF1 sequence of the M2 gene (Long strain) was amplified by PCR from the previously described plasmid L22K (García et al., 1993) and inserted into the pGEM-4 vector (pGEM/M2-1) under the control of a T7 promoter. A series of deletion mutants was constructed (Fig. 2a
) and used for transfection of HEp-2 cells previously infected with the vaccinia recombinant vTF-7.3 (Fuerst et al., 1986
). Expression of the mutant proteins was monitored by Western blotting with a polyclonal rabbit antiserum raised against purified 22K protein (serum
22K; Fig. 2b
). Specific bands that were absent in untransfected cells (Fig. 2b
, Mock) were observed in extracts of cells transfected with each of the plasmids. The electrophoretic mobility of these bands reflected the extent of the deletions. Differences in band intensity may reflect variability of transfection efficiencies with different plasmids, or different expression levels or stability of each mutant protein.
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A summary of the Western blot results obtained with all the mAbs is presented in Fig. 2(a), except for mAbs 22K2 and 22K6, which reacted poorly in this assay and whose pattern of reactivity could not be assigned unambiguously. Four reactivity patterns were discerned. The pattern observed with mAb 37M2 was reproduced with mAbs 22K3, 65 and 129. A second pattern was shared by mAbs 22K1, 22K4, 22K5 and 22K7. Finally, mAbs 312 and 112 each generated a unique reactivity pattern.
These results allowed us to locate tentatively the epitopes recognized by the different antibodies in the 22K protein primary structure (Fig. 2b). Since antibody reactivity was evaluated by Western blotting, we assumed that all epitopes were linear and that lack of reactivity of a given antibody with certain mutants indicated that some or all of the deleted amino acids were part of the epitope. With these considerations, epitope 37M2 (and 22K3, 65 and 129) was placed between residues 128 and 148 of the 22K polypeptide. Epitope 22K4 (and 22K1, 22K5 and 22K7) was located in the segment encompassing residues 148 to 166. Epitope 312 was located between residues 166 and 188. Finally, epitope 112 was placed between residues 2 and 6.
The results of Figs 1 and 2 led us to conclude that epitope 112 is buried in the 22K molecule but exposed under the denaturing conditions of Western blotting. Thus, we tried to refine the sequence requirements of epitope 112 and relate them to folding of the native 22K protein. Phosphorylation of the 22K protein is influenced not only by mutations at the sites of phosphorylation (Ser-58 and Ser-61) but also by changes in relevant amino acids distantly located in the protein primary structure (Hardy & Wertz, 2000
). Thus, phosphorylation can be used as a test for correct folding of the 22K protein.
The 24 kDa isoform of the 22K protein was expressed and labelled with [32P]orthophosphate in HEp-2 cells infected with vaccinia vTF7-3 and transfected with the plasmid pGEM/M2-1 (Fig. 3a, wt). Identity of this band was confirmed by immunoprecipitation using a pool of anti-22K mAbs (Fig. 3b
). In contrast, an analogous 32P-labelled band could not be detected in extracts from cells transfected with the plasmid carrying the
27 mutation either before (Fig. 3a
) or after (Fig. 3b
) immunoprecipitation. However, both proteins were expressed to comparable levels, as assessed by Western blotting with a pool of anti-22K mAbs (Fig. 3c
). Identity of the
27 mutant was confirmed by lack of reactivity with mAb 112 in a Western blot (Fig. 3d
). Thus, the
27 mutation prevented phosphorylation of the 22K protein, probably as a consequence of improper folding. In contrast to the
27 mutation, elimination of an equivalent number of amino acids at the C terminus (
C6) had no effect on expression or phosphorylation of the 22K protein (Fig. 3
).
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In conclusion, the results presented here indicate that epitope 112 is not exposed in the native 22K protein. Since most linear epitopes generally span six to ten contiguous amino acids, epitope 112 could include aa 56 to 1216 of the 22K polypeptide and one or two of the first cysteines in the Cys3-His1 motif. While the structure of this motif is not known for the 22K protein, the partial structure of the analogous motif in the transcriptional factor Nup475 has been determined by multidimensional nuclear magnetic resonance (Worthington et al., 1996). In this case, a zinc ion is coordinated by the three cysteines and the histidine. Interestingly, amino acids located N-terminal to the Cys3-His1 motif in Nup475 interact with the His residue, indicating a very compact local structure for this part of the protein. By analogy, the lack of reactivity of mAb 112 with the native 22K protein, and the influence of N-terminal amino acids (aa 46) in the folding of this molecule, could be explained by formation of a similar compact structure around the Cys3-His1 motif of the 22K protein.
Since all antibodies analysed in this study, with the exception of 112, recognized epitopes located in the C-terminal half of the 22K protein and they could immunoprecipitate the native molecule, it is likely that a substantial part of the C-terminal half of the 22K protein is exposed and thus accessible to antibodies. This is consistent with results reported by Tang et al. (2001) and Zhou et al. (2003)
indicating greater flexibility of the C-terminal half of the protein for sequence changes than the N-terminal 30 aa. The antibodies described here may prove valuable tools to study structural aspects of the 22K protein, interactions of this molecule with viral and cellular partners, and the mechanism of action of this transcription antiterminator.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 October 2004;
accepted 10 January 2005.
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