Characterizing Influenza NP Protein Interactions with Viral NS1 Factor

SMSU Event Center BC

Influenza virus rapidly evolves to evade vaccines and gain resistance to antiviral therapies. Yearly vaccination controls seasonal influenza, but pandemic prevention relies on effective antivirals. The viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is comprised of nucleoproteins (NP), the RNA genome segment, and the RNA dependent RNA polymerase (RdRP). NP is a highly conserved structural component of the vRNP, but also interacts with both viral and host factors to regulate viral RNA expression; making NP interactions a target for influenza inhibitors. NS1, a multifunctional viral protein whose primary role is to serve as an interferon antagonist, is reported to interact with NP. We aim to understand if the viral NS1 protein enhances viral RNA expression through interaction with NP. If so, this protein interaction represents a novel antiviral target. We examined reconstituted vRNPs, in the presence or absence of NS1, in Vero cells, which lack the host innate interferon antiviral response. Cells were collected and total RNA was isolated. RNA was resolved on 1% agarose gel to confirm RNA integrity, then reverse transcribed and quantitative PCR performed with specific primers. Our result confirms the addition of NS1 enhances RNA expression from reconstituted vRNPs in Vero cells. To directly evaluate NP-NS1 interaction we purified total protein from human tissue culture 293T cells and NP complexes were immunopurified and examined for NP and NS1 via western blot. NP interactions and vRNP activity will be examined with NS1 mutants. Our experiments will illuminate the role of NP-NS1 interaction.