E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors

Abstract

The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism-disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozinresistant strains. This work provides a new approach to design antiinfluenza drugs.T he RNA-dependent RNA polymerase (RDRP) of the influenza A virus is composed of polymerase basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA) (1). The function of RDRP for viral replication requires association with the nucleoprotein (NP) (2) to form the ribonucleoprotein (RNP) complex. Only low resolution structures of the RNP complex are available from cryo-EM studies (2-9), whereas high resolution structures have been reported for some individual components or fragments (10-12). Crystal structures of NP indicate that it exists in trimers (13,14), with the tail-loop (residues 402-428) region playing an important role in the trimerization (Fig. 1A). Based on the structural information, it was suggested that the tail-loop binding pocket could be a target for antiinfluenza therapeutics (13,14).Disruption of the NP-NP interaction as a strategy for designing antiinfluenza drugs has been further reported. Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers. These results support the importance of NP in the RDRP activity and viral replication, and the possibility of NP as a drug target. However, it remains to be shown that molecules capable of disrupting the NP-NP interaction would inhibit viral replication.Recently Kao et al. (19) and our group (20) reported the use of high throughput screening to identify nucleozin and its analogs as inhibitors that halt viral replication by binding to NP and causing it...

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein

“…In this report, N245-382 and N245-389 show a very high degree of stability compared to the other C-terminal domain truncations of the HCoV-229E N protein due to a high degree of subunit interactions. The inhibition of viral N protein oligomerization by developing competing peptides and small organic compounds is an attractive therapeutic strategy against viral infection [53][54][55]. We showed that a peptide based on the C-terminal tail interfered with the oligomerization of the C-terminal domain of the HCoV-229E N protein, N245-389 and performed the inhibitory effect on viral titre of HCoV-229E.…”

Structural Basis for RNA Binding and Homo-Oligomer Formation by Influenza B Virus Nucleoprotein

“…The identification of ligands which prevent the formation of the salt bridge will be of particular interest to combat both types of influenza viruses. Intriguingly, an inhibitor which targets the salt bridge of ANP has been recently identified (28). It will be interesting to find out whether this inhibitor also targets the BNP salt bridge.…”

Structure-Based Discovery of the Novel Antiviral Properties of Naproxen against the Nucleoprotein of Influenza A Virus

“…The swapping loop, important for the stabilization of the trimeric interface, was considered to be a good target for the design of new NPdirected antivirals based on structural studies (4,5). Indeed, the salt bridge between E339 and R416 from neighbor monomers required for NP trimerization was targeted by NP inhibitors identified by virtual screening (16). Other compounds targeting cellular proteins involved in the nuclear transport of the RNP or in signaling pathways exploited by influenza A virus also exhibited antiviral effects (17,18).…”