Human interactome of the influenza B virus NS1 protein.

Corinna Patzina,Catherine H Botting,Richard E Randall,Benjamin G Hale,Adolfo García-Sastre

doi:10.1099/jgv.0.000909

Abstract

NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.

Highlights

NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, interferon (IFN) induction
The NS1 proteins of both viruses are well described as multifunctional virulence factors that inhibit host interferon (IFN) production and IFN-induced antiviral effectors [3,4,5,6,7,8]
While the functions and host interactors of the FLUAV NS1 protein have been extensively characterized [9], far less is known about the properties of the FLUBV NS1 protein

Summary

IDENTIFICATION OF HUMAN PROTEINS

To identify human proteins interacting with B/NS1, including those potentially upregulated by IFN, we took advantage of the observation that HEp2-B/NS1 and HEp2-MCS cell lines responded normally to treatment with exogenous rIFNa, and optimized small-scale immunoprecipitation experiments. To validate a subset of these data, we confirmed the specific association of B/NS1 with DHX9, ILF3, YBX1 and HNRNPC by co-transfection of 293 T cells with GST or GST-tagged B/NS1 and the respective tagged host factor constructs [28,29,30], followed by GST pull-down and identification of co-precipitated proteins by western blot (Fig. 2d– g) These four factors are all known RNA-binding proteins, and by performing the pull-downs either in the absence or presence of RNase A we were able to show that B/NS1 does not appear to interact indirectly with ILF3 or HNRNPC via common RNA-binding activities (Fig. 2e and g).

Input myc GST

CONCLUDING REMARKS