Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication

Abstract

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.

Figure 1. Virus type-specific conservation of the PA-binding domain and interaction of PA with PB1.

(A) Upper panel: Alignment of the N-terminal 25 aa of FluA and FluB PB1. The dotted box indicates the 3₁₀-helix comprising the core PA-binding domain of PB1. FluA-specific (blue) and FluB-specific (red) aa are highlighted. Middle and lower panels: Alignment of the N-terminal 25 aa of all available FluA and FluB sequences available in the NCBI influenza virus database. Figures on the right hand side indicate the number of sequences present in the database. Grey bars highlight aa which reconstitute the 3₁₀-helix of FluA PA and possibly of FluB PA. (B) A/SC35M- and B/Yamagata/73-derived PB1 chimeras used in (b). Note that all PB1 proteins were expressed with C-terminal HA-tags. (C) Human 293T cells were transfected with expression plasmids coding for the indicated PB1 proteins and the C-terminally hexahistidine-tagged PA of FluA (FluA-PA_His). Cell lysates were prepared 24 hours post transfection and subjected to immunoprecipitation (IP) using anti-HA (αHA) agarose. Precipitated material was separated by SDS-PAGE and analyzed by Western blot for the presence of either His- or HA-tagged polymerase subunits using appropriate antibodies. Protein expression was controlled by analyzing equal amounts of cell lysate. Molecular weights are shown in kilodaltons.

Figure 2. Quantification of the interaction between PB1_1–25 and PA.

(A) Determination of the 50% inhibitory concentration (IC₅₀) of PB1_1–25A by competitive ELISA using the indicated increasing concentrations of peptides and cell extract containing HA-tagged PA of FluA. Error bars represent standard deviations from triplicate experiments. (B) IC50 of PB1_1–25A-derived peptides. S.D. is shown in parenthesis. Asterisks indicate highest concentrations of peptides (3000 nM) used without detectable inhibitory effect. Grey boxes highlight amino acids that are part of the 3₁₀-helix, which was postulated to comprise the core PA-binding region of PB1. Amino acids known to form hydrogen bonds with PA residues are represented in bold.

Figure 3. Binding characteristics of FluA and FluB PB1-derived peptide chimera.

Inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA. Competitor peptides (0.048 to 3000 nM) were mixed with cell extracts containing HA-tagged PA from either FluA or FluB. Letters in red indicate FluB, letters in blue FluA specific aa. S.D. is indicated in parenthesis. Asterisks indicate highest concentrations of peptides used without reaching 50% inhibition.

Figure 4. Dual-binding properties of the FluA/B peptide chimera PB1_1–25A_T6Y.

(A) – (D) Binding of overexpressed HA-tagged PA subunits of differing influenza strains in cell extracts to the immobilized peptides corresponding to the domains of FluA PB1 (PB1_1–25A), FluB PB1 (PB1_1–25B) or FluA PB1 T6Y (PB1_1–25A_T6Y) was determined by ELISA. Signals using the cognate peptide and lysate were normalized to 1. Binding of the PA subunits to the control peptides was not observed (data not shown). Upper panels: Western blot of the PA-containing cell extracts used. Molecular weights shown in kilodaltons. (F) Structure of FluA PB1_1–15 (green) bound to FluA PA (grey) as published . T6 forms a hydrogen bond (green line) to a water molecule (blue). Molecular modeling suggests that the aromatic side chain in the mutant T6Y (orange) fits into a hydrophobic pocket and displaces the water molecule.

Figure 5. Virus-type independent binding and inhibition of GFP fused to the dual binding peptide PB1_1–25A_T6Y.

(A) GFP-PB1 fusion proteins used in (B). (B) Complex formation of PB1_1–25-derived GFP fusion proteins and HA-tagged PA of FluA and FluB. Indicated proteins were expressed in human 293T cells and binding of the GFP fusion proteins was analyzed by IP using anti-HA agarose and subsequent immunoblotting. Precipitated material was visualized using the indicated antibodies for the presence of either HA-tagged PA or GFP. Molecular weights are shown in kilodaltons. (C) Polymerase inhibitory activity of PB1_1–25-derived GFP fusion proteins in FluA and FluB polymerase reconstitution assays. 293T cells were transiently transfected with a plasmid mixture containing either Flu A (A/WSN/33) or Flu B (B/Yamagata/73) PB1-, PB2-, PA- and NP-expression plasmids, polymerase I (Pol 1)-expression plasmid expressing an influenza virus-like RNA coding for the reporter protein firefly luciferase (FluA) or (FluB) to monitor viral polymerase activity and expression plasmids coding for the indicated GFP fusion proteins. The transfection mixture also contained a plasmid constitutively expressing renilla luciferase (100 ng), which served to normalize variation in transfection efficiency. The activity observed with transfection reactions containing Flag-GFP were set to 100%. The omission of PB2 in the transfection mixture served as a negative control.

Figure 6. Inhibition of FluA and FluB by the dual binding peptide PB1_1–25A_T6Y.

Plaque reduction assay using PB1_1–25A-Tat; PB1_1–25A_T6Y-Tat; PX-Tat (control peptide) with FluA, FluB and VSV (vesicular stomatitis virus). A H₂O control was used to standardize the assay to 100%. Note that PB1_1–25B-Tat could not be tested due to insolubility. Error bars represent S.D.