Abstract
Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.
Highlights
Influenza is an infectious disease of birds and mammals caused by the influenza viruses belonging to the family Orthomyxoviridae[1]
Understanding structural and mechanistic information regarding influenza A virus NP and its interactions with RNA should facilitate the discovery of agents that block the formation of ribonucleoprotein (RNP) during viral genome replication
Li et al reported that influenza A virus with NP mutations in Y148, R150, R152, R156, R174, R175, R195, R199, R213, and R221 were significantly attenuated[16]
Summary
Influenza is an infectious disease of birds and mammals caused by the influenza viruses belonging to the family Orthomyxoviridae[1]. The viral surface proteins hemagglutinin and neuraminidase have played important roles in antiviral drug discoveries and provide crucial neutralization against the virus[3]. Influenza A virus nucleoprotein (NP) is a major virion structural protein that has been predicted to interact with negative-strand viral RNA during viral nucleocapsid formation[8]. Several residues that are critical for RNA binding and virus infectivity in the influenza A virus NP have been identified[16,17]. Understanding structural and mechanistic information regarding influenza A virus NP and its interactions with RNA should facilitate the discovery of agents that block the formation of ribonucleoprotein (RNP) during viral genome replication. By targeting Y148, we identified an influenza virus NP inhibitor, H7 [(E,E) -1,7-bis(4-hydroxy-3-me thoxyphenyl)-1,6-heptadiene-3,5-dione], which reduced the NP’s RNA-binding affinity and hindered viral replication. We present a structural model of the influenza NP in complex with RNA, which clearly illustrates the critical role of Y148
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