Structure-based design of NS2 mutants for attenuated influenza A virus vaccines.
We previously characterised the matrix 1 (M1)-binding domain of the influenza A virus NS2/nuclear export protein (NEP), reporting a critical role for the tryptophan (W78) residue that is surrounded by a cluster of glutamate residues in the C-terminal region that interacts with the M1 protein (Akarsu et al., 2003). To gain further insight into the functional role of this interaction, here we used reverse genetics to generate a series of A/WSN/33 (H1N1)-based NS2/NEP mutants for W78 or the C-terminal glutamate residues and assessed their effect on virus growth. We found that simultaneous mutations at three positions (E67S/E74S/E75S) of NS2/NEP were important for inhibition of influenza viral polymerase activity, although the W78S mutant and other glutamate mutants with single substitutions were not. In addition, double and triple substitutions in the NS2/NEP glutamine residues, which resulted in the addition of seven amino acids to the C-terminus of NS1 due to gene overlapping, resulted in virus attenuation in mice. Animal studies with this mutant suggest a potential benefit to incorporating these NS mutations into live vaccines.
Akarsu
Hatice
H
Unit of Virus Host-Cell Interactions, UMI 3265, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9, France.
Iwatsuki-Horimoto
Kiyoko
K
Noda
Takeshi
T
Kawakami
Eiryo
E
Katsura
Hiroaki
H
Baudin
Florence
F
Horimoto
Taisuke
T
Kawaoka
Yoshihiro
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D004285:Dogs; D005260:Female; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity*; D007252:Influenza Vaccines / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009976:Orthomyxoviridae Infections / Q000821:virology; D016019:Survival Analysis; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism
D007252:Influenza Vaccines; D050505:Mutant Proteins; C556232:NS2 protein, influenza virus A; D014613:Vaccines, Attenuated; D017361:Viral Nonstructural Proteins