A peptide derived from the C‐terminus of PB1 inhibits influenza virus replication by interfering with viral polymerase assembly

Abstract

Efficient assembly of the influenza virus RNA-dependent RNA polymerase, a heterotrimeric complex formed by three subunits (PA, PB1 and PB2) is critical for virus replication and pathogenicity. Therefore, interfering with the assembly of the RNA-dependent RNA polymerase complex could offer novel and effective anti-influenza therapeutics. In the present study, we show that a short peptide derived from amino acids 731-757 of PB1 (PB1 ) can disrupt the interaction between the C-terminal part of PB1 (denoted as PB1c corresponding to PB1 ) and the N-terminal part of PB2 (denoted as PB2n corresponding to PB2 1-40 ). We further show that PB1 731-757 is capable of inhibiting viral polymerase activity and viral replication. Interestingly, we find that PB1 731-757 interacts with PB1c rather than PB2n. Furthermore, mutational analyses show that the hydrophobic sites of PB1c play an essential role in the PB1c-PB1 731-757 interaction. The characterization of the inhibitory effect of PB1 731-757 on viral polymerase activity and viral replication could offer a potential target for anti-influenza drug development. Structured digital abstract

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System

“…It was the same with PB1 731-757 but not with its I750N mutants. The results were correlated with a previous report that PB1 inhibited the PB1c-PB2n association by binding to PB1c whereas a PB1 731-757 I750N mutant did not (12). The results indicated that the Tet on-BiLC assay can be used to screen inhibitors of the PB1-PB2 association.…”

“…Sugiyama et al found that PB1c-PB2n interaction was mainly mediated by 2 residues on the hydrophobic patch of PB1c (39), although there may have been other regions on PB1 or PB2 that contributed to the PB1-PB2 interaction (10,11,40). And our previous studies showed that PB1 bound to the hydrophobic patch of PB1c to inhibit influenza virus replication (12). These results indicated that the hydrophobic patch formed by PB1c could be a potential drug target for screening inhibitors.…”

“…Inhibitors such as PB1 and PB1 have been shown to suppress different strains of influenza virus (12,43), indicating that inhibitory compounds targeting the interaction surface of influenza virus polymerase likely have broad efficacy. Here PB1c and PB2n were used to mimic the PB1-PB2 interaction since the PB1c-PB2n interaction mainly mediates the PB1-PB2 interaction and, more importantly, the PB1c-PB2n interaction is much stronger than the PB1-PB2 interaction ( Fig.…”

“…In order to investigate the specificity inhibitory function of molecule 5 with respect to the PB1c-PB2n interaction, its effects on PB1-PB2 and PA-PB1 interactions and even heterotrimer assembly were further detected by our BiLC assay. Since previous studies found that PB1 1-25 could suppress the PA-PB1 interaction by interacting with PA and that PB1 731-757 could suppress the PB1-PB2 interaction by binding to PB1c (12,(43)(44)(45), PB1 1-25 and PB1 731-757 were used as controls to test whether the BiLC method could be used to illustrate and identify potential inhibitors that target influenza virus polymerase assembly. First, PB1 and PB1 731-757 were cloned onto the N terminus of GFP, which facilitates the expression of the peptide.…”

“…The RNA-dependent RNA polymerase (RdRp) complex of influenza virus, responsible for RNA synthesis, is a heterotrimeric complex composed of three subunits-PA, PB1, and PB2 (7). Since the structure and function relationships of influenza virus polymerase have been well illustrated, the protein-protein interactions (PPIs) between influenza virus polymerase subunits have been shown to be potential drug targets for structure-based drug design (8)(9)(10)(11)(12).…”