University of Warwick, Department of Biological Sciences, Coventry CV4 7AL, UK
Correspondence
Tony Marriott
a.c.marriott{at}warwick.ac.uk
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ABSTRACT |
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Present address: Department of Pathology & Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
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MAIN TEXT |
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The N protein can be assayed for its function in RNA replication, transcription and encapsidation using a minigenome replication assay, in which plasmids encoding the minigenome and the N, P, L and M2-1 proteins are transfected into an appropriate cell line (Marriott & Easton, 1999). An attempt to map functionally important regions on the bRSV N protein by means of deletions found that all internal deletions resulted in inactivity, and deletion of as little as two residues from the N terminus or one residue from the C terminus was sufficient to reduce activity by 9598 % (Khattar et al., 2000
). This suggests that the structural integrity of the N protein is compromised by deletions, resulting in a loss of the active conformation. An alternative approach to deletion mapping of a protein is to substitute cognate regions from a homologous protein to generate a chimeric protein, which is much more likely to retain an active conformation than a deleted form of the protein. In this report we describe the use of chimeric proteins to identify a region of the RSV N protein essential for its template activity, as well as binding to the P protein.
Pneumonia virus of mice (PVM), a member of the genus Pneumovirus, causes a severe respiratory infection in laboratory mouse colonies (Horsfall & Hahn, 1939, 1940
). PVM, or a serologically related virus, is also widespread in the human population (Pringle & Eglin, 1986
). The N protein of PVM shows 60 % identity to the RSV N protein, with a highly conserved region (residues 245315) showing 96 % identity (Barr et al., 1991
) (Fig. 1
). Analysis of deletion mutants of the PVM N protein suggested that a region between residues 309 and 381 was important in binding to the PVM P protein (Barr & Easton, 1995
). A series of chimeric plasmids were constructed in which the C-terminal part of the RSV N gene ORF was replaced with the equivalent region of the PVM N gene ORF. Joins were in regions of exact identity between the protein sequences in order to maintain protein integrity (Fig. 1
, underlined) and were achieved by ligating together PCR products with HgaI restriction sites at the position of the join (HgaI is an enzyme that cleaves to one side of its recognition site such that the recognition site is removed from the ligated product). Details of plasmid constructions are available from the authors upon request. Western blot analysis showed that each plasmid directed production of a protein of the expected size, 43 kDa, when transfected into cells expressing T7 RNA polymerase (data not shown).
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The bRSV N protein retains the ability to co-precipitate P protein and to encapsidate RNA, with the C-terminal 27 residues deleted, despite being inactive in replication (Khattar et al., 2000). Deletion of a further single amino acid abolished both P protein binding and RNA encapsidation. This suggests that residues 365391 are dispensable for P protein binding. If this is applicable to human RSV N protein, this would further delineate the residues critical for binding to P protein, to A352, Q356, E359 and G361.
The ability of the N protein to encapsidate RNA into nuclease-resistant nucleocapsids was not addressed directly in this study. Encapsidation appears to be relatively non-specific in terms of RNA sequence (Marriott et al., 2001) and cellular RNA is encapsidated in the absence of other viral proteins (Meric et al., 1994
). By analogy with Sendai virus, it is hypothesized that the P protein enables the N protein to encapsidate the viral genome specifically (Curran et al., 1995
). Chimeric RPN374 N protein is clearly able to encapsidate and protect the viral minigenome (Fig. 2b
, lane 4), but encapsidation in the absence of replication was below the level of reliable detection in our blots. However, a longer exposure of the blot in Fig. 2(b)
did suggest that the RPN223 protein is also able to encapsidate RNA (Fig. 2c
). The smear of hybridizing material seen in Fig. 2(c, lane 8) may represent partial loss of nuclease-resistance by the RPN293 protein.
Loss of ability of the N protein to support replication in this study maps to the same six amino acid changes as loss of binding to the P protein, suggesting that the NP interaction is more important to RSV replication than the NRNA interaction. Although this hypothesis requires further investigation, this would imply that reagents able to interfere with NP binding may be highly inhibitory to RSV replication and thus be potential therapeutic reagents for RSV respiratory disease.
We suggest that the strategy of producing chimeric proteins from homologous, structurally related proteins is a useful one for mapping functional residues or regions on a protein, which is less likely to be affected by incorrect folding than the traditional deletion approach. We, and others, have applied the chimeric protein approach recently to analyse the functional domains of the RSV M2-1 transcriptional enhancer protein (unpublished data; Zhou et al., 2003).
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ACKNOWLEDGEMENTS |
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Received 23 May 2003;
accepted 27 June 2003.
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