Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase

Abstract

The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

Influenza Virus Adaptation PB2-627K Modulates Nucleocapsid Inhibition by the Pathogen Sensor RIG-I

“…This would implicate that the strength of the polymerase -nucleocapsid interaction influences RIG-I activity. To directly test this hypothesis, we disrupted the polymerase complex with a PB1-derived peptide (PB1-T6Y) (Wunderlich et al, 2011). The inhibitor peptide massively increased RIG-I activation by incoming nucleocapsids, and independent of the PB2-627 signature (Figures 6D and S6C).…”

“…Then, cells were treated with 10 ng/ml Borna-X-Tat or PB1 1-15 T6Y-Tat (Wunderlich et al, 2011) dissolved in medium with 0,2% BSA. At 1 h post-treatment, cells were lysed and subjected to the RIG-I conformational switch assay.…”

Identification of Host Genes Linked with the Survivability of Chickens Infected with Recombinant Viruses Possessing H5N1 Surface Antigens from a Highly Pathogenic Avian Influenza Virus

“…The amino acid at aa 670 in PA and that between LP and WB were identical; therefore, substitution at aa 14 in PB1 was thought to affect the hydrophobic bond between PA and PB1. Wunderlich et al reported that substitution from alanine to valine at aa 14 of PB1 increased binding between PB1 and PA (49). As PB1 of LP possesses valine at aa 14 while that of WB is alanine, PB1 of LP could contribute to increasing the binding between PB1 and PA, thus contributing to the decreased survivability.…”

Targeting of the Influenza A Virus Polymerase PB1-PB2 Interface Indicates Strain-Specific Assembly Differences

“…We recently showed that assembly inhibition by a peptide derived from the PA interaction domain of PB1 (PB1 ) can interfere with viral replication of several influenza A viruses (16,43). Furthermore, peptide screenings revealed amino acid substitutions which enhanced the affinity of PB1 for the PA protein and led to a more efficient inhibition of viral growth (42,43). To address whether the PB1-PB2 interaction site provides a similar potential for assembly inhibitors, we fused the PB1-binding domain of PB2 (PB2 ) to green fluorescent protein (PB2 1-37 -GFP) to facilitate stability and detection and tested this fusion construct for inhibition of influenza A virus polymerase activity in polymerase reconstitution assays.…”