Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

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.

Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates

“…Due to differences in the way many viruses replicate, and in some cases due to virus family-centric literature, many of these proteins could only be classified as functionally equivalent HIV-1 Gag and NC domain orthologs from their disorder and Zn 2+ -dependence, or from their propensity to multimerize, phase-separate, and bind vRNAs [ 55 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 ]. Other orthologous selected proteins including non-structural proteins are previously characterized as being disordered early replication intermediates that bind vRNAs for assembly and encapsidation, or as providing structure, stability, resistance, and infectivity to virus cores [ 49 , 75 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ]. Finally, other informative reports have described some selected orthologous viral proteins as disordered vRNA or nucleoprotein chaperones [ 55 , 105 , 106 , 107 , 108 ].…”

Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions

“…Based on this, we speculate that NP7 and NP7-R31G change the RNA binding properties of NP and thereby influence the function of multiple packaging sequences to establish RNA-RNA interactions between the eight vRNPs. Indeed, within the vRNP structure (11), several NP7 amino acid residues are located close to the putative RNA binding groove (35,36) (Fig. 4L).…”

Naproxen Exhibits Broad Anti-influenza Virus Activity in Mice by Impeding Viral Nucleoprotein Nuclear Export

“…Meanwhile, the nuclear export of NP and vRNP complexes has been reported to be inhibited by the compounds RK424 (Kakisaka et al, 2015), Amaryllidaceae alkaloid (He et al, 2013), PPQ-581 (Lin et al, 2015), (Perwitasari et al, 2014), and KR-23502 (Jang et al, 2016). Furthermore, the self-oligomerization and RNA-binding capacities of NP were shown to be suppressed by naproxen (Lejal et al, 2013), H7 (Liu et al, 2016), and 3-mercapto-1,2,4-triazole (Liu et al, 2016). Among these compounds, nucleozin is the only inhibitor for which the antiviral mechanism is well documented.…”