Association of influenza virus matrix protein with ribonucleoproteins.
To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.
Ye
Z
Z
Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA. yez@cber.fda.gov
Liu
T
T
Offringa
D P
DP
McInnis
J
J
Levandowski
R A
RA
D000818:Animals; D001665:Binding Sites; D002642:Chick Embryo; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; D012261:Ribonucleoproteins; D014763:Viral Matrix Proteins; D012313:RNA