A monoclonal antibody binds to threonine 49 in the non-structural 1 protein of influenza A virus and interferes with its ability to modulate viral replication

Abstract

The emergence of resistant influenza A viruses highlights the continuous requirement of new antiviral drugs that can treat the viral infection. Non-structural 1 (NS1) protein, an indispensable component for efficient virus replication, can be used as a potential target for generating new antiviral agents. Here, we study the interaction of 2H6 monoclonal antibody with NS1 protein and also determine whether influenza virus replication can be inhibited by blocking NS1. The 2H6-antigen binding fragment (Fab) forms a multimeric complex with the NS1 RNA-binding domain (RBD). T49, a residue which forms a direct hydrogen bond with double stranded RNA, in NS1 protein was found to be critical for its interaction with 2H6 antibody. NS1(RBD) has high affinity to 2H6 with KD of 43.5±4.24nM whereas NS1(RBD)-T49A has more than 250 times lower affinity towards 2H6. Interestingly, the intracellular expression of 2H6-single-chain variable fragment (scFv) in mammalian cells caused a reduction in viral growth and the M1 viral protein level was significantly reduced in 2H6-scFv transfected cells in comparison to vector transfected cells at 12h post infection. These results indicate that the tight binding of 2H6 to NS1 could lead to reduction in viral replication and release of progeny virus. In future, 2H6 antibody in combination with other neutralizing antibodies can be used to increase the potency of viral inhibition.

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

“…These predictions are in agreement with the results shown in Fig. 2 and in our previous publication 29 . However, this model also predicted that R44 of NS1(RBD) could be involved in the interaction with 2H6-Fab because it was in close proximity to two residues in VL-CDR1.…”

“…This was conducted by using HADDOCK on 192 water-refined models, including an analysis of energy contributions from Van der Waals interaction, electrostatic interaction, restraints violation and buried surface area 32 . As comparative ELISA in this and previous studies 29 showed that residues N48 and T49 in NS1(RBD) are important for the interaction with mAb 2H6, they were defined as active residues involved in the binding interaction to generate a series of models of the NS1(RBD) and 2H6-Fab complex.…”

“…Since the NS1 protein is an intracellularly expressed viral protein, which is subjected to less host selective immune response and thus has lower mutation rate, antibodies targeting this protein could be helpful for the development of therapeutic treatment of influenza A infection. Indeed, our previous study showed that mAb 2H6 binds to the highly conserved T49 residue in NS1 and reduces viral replication of H1N1-PR8 in mammalian cell lines 29 .…”

“…In our previous study, we used full-length NS1 protein of H5N1 IAV to generate a monoclonal antibody (mAb) 2H6 and found that it could cross-react with NS1 of H3N2 and H1N1 subtypes 28 . MAb 2H6 binds to the RBD of NS1(RBD) and the intracellular expression of 2H6-single-chain variable fragment (scFv) in mammalian cells reduced the replication of A/Puerto Rico/8/1934(H1N1) (H1N1-PR8) virus 29 . In the present study, we used biochemical, structural and modelling methods to define the molecular interface mediating the interaction between mAb 2H6 and NS1.…”

Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks

“…Some of the identified phosphorylation sites occur at sites conserved between various IAV strains with a potential functional relevance. For example, we identified an NS1 modification at Thr49, which is immediately adjacent to the previously identified phosphorylation site at Ser48 in a region that is directly involved in RNA binding and therefore functionally relevant (47,49,50). In order to obtain insight into the structural consequences of Ser48/Thr49 phosphorylation, published NS1 structures (51, 52) were used as the templates to model the structure of SC35M NS1 using the Swiss-Model server (Fig.…”