Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody

Abstract

We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.

A NS1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza A virus

“…4B, single mutation at either residue P85 or Y89 of NS1 did not affect the rate of viral protein synthesis in single-cycle replication, since the expression levels of viral NP in mutant viruses were comparable to that of the wild-type virus at both time points. Similarly, the level of NS1, as determined by using a rabbit anti-NS1 polyclonal antibody (Wu et al 2016), was also comparable for all four viruses. However, mAb 19H9 only bound strongly to rgPR8-NS1-WT virus and did not bind the three mutant viruses.…”

“…Commercial antibodies against p85β (Santa Cruz), GST (Santa Cruz), GAPDH (Santa Cruz), Myc (Santa Cruz) and NP (Millipore) were used. A home-made rabbit anti-NS1 polyclonal antibody (Wu et al 2016) as well as the mAb 19H9 generated above were also used.…”

“…(B), Western blot analysis of 293T cell lysates infected with recombinant viruses at an MOI of 1. Cell lysates were collected at 12 and 24 hpi and subjected to Western blot analysis using mAb 19H9, anti-NP, anti-GAPDH, anti-NS1 (Wu et al 2016) antibodies. The experiments were repeated three times independently and a representative set of data is shown.…”

Generation and Characterization of the Monoclonal Antibody Panel Specific to the NS1 Protein of the Influenza A Virus

“…It was demonstrated in model experiments that the interaction between antibodies and certain amino acid residues in the effector domain of the NS1 protein hinders NS1 interaction with double-stranded RNA [7] or cellular components, such as CPSF30 and eIF4G1 (Eukaryotic Translation Initiation Factor 4 Gamma I), which play an important role in mRNA processing [8], thereby significantly inhibiting the replication of the influenza A viruses.…”

“…Monoclonal antibodies (MAbs) may serve as an efficient tool in such studies. MAbs are successfully used to map NS1 epitopes and to characterize the polymorphism of its structure, as well as to study the functions of this protein [7,[9][10][11].…”