Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities.
Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.
Chan
Wai-Hon
WH
Department of Biochemistry, the Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.
Ng
Andy Ka-Leung
AK
Robb
Nicole C
NC
Lam
Mandy Ka-Han
MK
Chan
Paul Kay-Sheung
PK
Au
Shannon Wing-Ngor
SW
Wang
Jia-Huai
JH
Fodor
Ervin
E
Shaw
Pang-Chui
PC
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019590:Nucleocapsid Proteins; D011487:Protein Conformation; D055503:Protein Multimerization; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins
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