Influenza A virus causes annual epidemics and is potential threat for widespread pandemics. The genome of influenza A virus consists of eight segments of (-)RNA that are encapsidated in distinct double-helical structures called the ribonucleoprotein (RNP) complexes. The nucleoprotein (NP), the major protein component of RNPs, binds along genomic RNA at a 24nt interval. The viral polymerase, consisting of PA, PB1, and PB2, is bound to the two RNA termini of the RNP. NP is abundantly made in infected cells, and is essential for important viral processes such as viral genome trafficking, viral RNA replication, and virus assembly. Our laboratory has recently determined the crystal structure of influenza A virus NP, which shows an overall fold and an external RNA binding mode different from those of rhabdoviruses. Several key residues for RNA binding have been identified by mutagenesis. Polarization anisotropy assay shows that wild-type NP exhibits strong binding affinity for RNA, which in turn stimulates NP self-oligomerization. Without RNA, NP oligomers are not stable and slowly dissociate to monomers. Mutant NP monomer that cannot oligomerize shows weak RNA binding, suggesting that either RNA binds to NP-NP interface or NP monomer has a tertiary structure different from oligomeric NP. Purified NP can be phosphorylated at S402/S403 in vitro, and phosphorylation reduced NP RNA binding. Using purified recombinant NP and viral polymerase, we have shown that NP stimulates unprimed viral RNA synthesis by interacting directly with the polymerase.
Our specific aims are to: 1. Elucidate the linked mechanisms of NP RNA-binding, oligomerization and assembly into RNPs. We will: (1) determine the structure of the NP monomer to elucidate the mechanism of NP monomer?oligomer transition;(2) determine the RNA binding mode of NP using site-directed mutagenesis and fluorescence anisotropy;(3) solve the structure of the NP:RNA complex by X-ray crystallography and cryo-EM reconstruction to obtain an accurate atomic model for the structure of the RNP;(4) elucidate the effects of phosphorylation of specific sites of NP on its various functions;and (5) solve the structure of NP from infectious salmon anemia virus (ISAV) and influenza B virus and determine whether NP self-oligomerization and RNA binding modes are broadly conserved in the Orthomyxoviridae family. 2. Elucidate the role of NP in viral RNA replication. To understand how NP interacts with the polymerase and affects RNA synthesis, we will: (1) identify the polymerase PA sequence(s) that regulate NP-directed unprimed viral RNA replication;(2) identify the sites on NP that functionally interact with the polymerase;and (3) use our in vitro system to elucidate several key issues concerning NP-dependent viral RNA replication. Our proposed research employs a broad spectrum of X-ray crystallography, electron microscopy and other biophysical techniques (by Dr. Tao, PI) together with functional and virological methods (by Dr. Krug, co-PI). Results from our studies will have important applications in antiviral drug development.

Public Health Relevance

Influenza viruses cause highly contagious, acute respiratory illnesses and pose a serious threat to the human public health. The influenza virus nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complex, and plays critical roles in viral RNA replication.
The aim of this grant is to elucidate the molecular mechanism and biological significance of several key activities of the influenza A virus nucleoprotein and to discover how such activities can be exploited for antiviral development.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Hauguel, Teresa M
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Rice University
Schools of Arts and Sciences
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Zheng, Wenjie; Olson, John; Vakharia, Vikram et al. (2013) The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein. PLoS Pathog 9:e1003624
Zheng, Wenjie; Tao, Yizhi Jane (2013) Structure and assembly of the influenza A virus ribonucleoprotein complex. FEBS Lett 587:1206-14
Guu, Tom S Y; Zheng, Wenjie; Tao, Yizhi J (2012) Bunyavirus: structure and replication. Adv Exp Med Biol 726:245-66
Ye, Qiaozhen; Guu, Tom S Y; Mata, Douglas A et al. (2012) Biochemical and structural evidence in support of a coherent model for the formation of the double-helical influenza A virus ribonucleoprotein. MBio 4:e00467-12
Marklund, Jesper K; Ye, Qiaozhen; Dong, Jinhui et al. (2012) Sequence in the influenza A virus nucleoprotein required for viral polymerase binding and RNA synthesis. J Virol 86:7292-7