Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein.
The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.
Li
Zejun
Z
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Watanabe
Tokiko
T
Hatta
Masato
M
Watanabe
Shinji
S
Nanbo
Asuka
A
Ozawa
Makoto
M
Kakugawa
Satoshi
S
Shimojima
Masayuki
M
Yamada
Shinya
S
Neumann
Gabriele
G
Kawaoka
Yoshihiro
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004285:Dogs; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019590:Nucleocapsid Proteins; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D000596:Amino Acids; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins