Previous Article | Next Article ![]()
Journal of Virology, September 1999, p. 7357-7367, Vol. 73, No. 9
Division of Virology, Department of
Pathology, University of Cambridge, Cambridge CB2 1QP, United
Kingdom
Received 22 March 1999/Accepted 25 May 1999
The influenza virus nucleoprotein (NP) is a
single-strand-RNA-binding protein associated with genome and antigenome
RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by
which NP binds RNA, we undertook a physical and mutational analysis of
the polypeptide, with the strategy of identifying first the regions in
direct contact with RNA, then the classes of amino acids involved, and
finally the crucial residues by mutagenesis. Chemical fragmentation and
amino acid sequencing of NP that had been UV cross linked to
radiolabelled RNA showed that protein-RNA contacts occur throughout the
length of the polypeptide. Chemical modification experiments implicated
arginine but not lysine residues as important for RNA binding, while
RNA-dependent changes in the intrinsic fluorescence spectrum of NP
suggested the involvement of tryptophan residues. Supporting these
observations, single-codon mutagenesis identified five tryptophan, one
phenylalanine, and two arginine residues as essential for high-affinity
RNA binding at physiological temperature. In addition, mutants unable
to bind RNA in vitro were unable to support virus gene expression in
vivo. The mutationally sensitive residues are not localized to any
particular region of NP but instead are distributed throughout the
protein. Overall, these data are inconsistent with previous models
suggesting that the NP-RNA interaction is mediated by a discrete
N-terminal domain. Instead, we propose that high-affinity binding of
RNA by NP requires the concerted interaction of multiple regions of the
protein with RNA and that the individual protein-RNA contacts are
mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Amino Acid Residues of Influenza
Virus Nucleoprotein Essential for RNA Binding
*
Corresponding author. Mailing address: Division of
Virology, Department of Pathology, University of Cambridge, Tennis
Court Rd., Cambridge CB2 1QP, United Kingdom. Phone: 44 1223 336918. Fax: 44 1223 336926. E-mail:
pd1{at}mole.bio.cam.ac.uk.
This article has been cited by other articles:
| J. Bacteriol. | Mol. Cell. Biol. | Microbiol. Mol. Biol. Rev. |
|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|