Alanine-Scanning Mutagenesis for Revealing the Role of Highly Conserved Regions of Influenza A Virus Neuraminidase

Abstract

Background: Neuraminidase (NA), a surface glycoprotein of influenza A virus, is an important molecular target for antiviral drugs. Recent reported NA-inhibition drug resistant cases, such as mutations in H274Y in N1 and R292K and E119G/A/D in N9 and N2, however, has raised concerns about the needs for better understanding NA functional role towards developing a new anti-influenza drug. Methods: Twenty-eight highly conserved amino acid residues in influenza A viral NA protein were identified through in silico analysis based on 2,827 NA sequences deposited in GenBank, NCBI. To understand the role of conserved residues on viral viability, we have introduced series of mutations (alanine substitutions) into NA by reverse genetics using A/WSN/33(H1N1) as a backbone. Results: Seven out of 28 mutants were rescued, indicating that the other 21 positions in NA are essential to viral viability. Among those 21 lethal mutants, 5 were rescued by exogenously adding NA from C. perfringens, suggesting that these 5 positions may reside on the NA active site. This assumption was reinforced by structural modeling by SWISS-MODEL server. By simulation, we also found 9/21 mutants located on the side-surface of NA protein. When being substituted, they remarkably reduced the virus survival by losing its biological function, possibly due to the associated structural alteration. Conclusion: This study identified several amino acid residues important for viral viability. Via structural simulation they are found located at NA active sites or oligomerization sites. We believe the results obtained herein provide valuable information in antiviral drug design targeting influenza NA protein. Mok, C.K., et al. 2008 Chang Gung University 2 Table of