OSR Journal of Student Research

Article Title

Characterizing NP mutant interactions to reveal novel antiviral targets


Influenza A viruses cause widespread outbreaks of respiratory infections that are life-threatening. There is a critical need for novel antiviral therapies due to the emergence of strains resistant to current antiviral medications. The nucleoprotein (NP), along with the viral RNA dependent RNA polymerase (RdRP) and RNA genome the viral ribonucleoprotein (vRNP), is responsible for influenza RNA synthesis. NP is highly conserved among influenza subtypes, making NP an excellent antiviral target. Here, we examine NP interactions with viral PB2, a subunit of the RdRP. Along with wild type NP, NPbd3, a NP mutant with glycine substitutions at 5 amino acids in an accessible and conserved site within the NP body domain, were analyzed for co-sedimentation of NP and PB2. Human tissue culture 293T cells were transfected with wild type NP or NP mutant alone and in combination with PB2.Total protein extracts were separated on a sucrose gradient. After ultracentrifugation, fractions were collected and analyzed by Western blot. Multiple trials confirm wild type NP co-sediments with PB2 while NPbd3 does not. This data supports our hypothesis that NPbd3 is non-functional for vRNP activities because its interaction with PB2 is disrupted. We aim to examine double glycine substitutions within this NP body domain to characterize NP-PB2 interaction further. NP with glycine substitutions at just two amino acids, 308/309, demonstrate very reduced vRNP activity; these residues are implicated in binding to the small molecule inhibitor nucleozin. Our research will better characterize NP interactions as a target for development of novel antivirals to combat Influenza.

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