Presentation Title

The Effect of Nucleozin on Influenza A Nucleoprotein Interactions

Faculty Mentor

Laura Newcomb

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

94

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Influenza A viruses cause seasonal epidemics of respiratory illness and are responsible for health and economic burdens annually. The virus is controlled with annual vaccines, which have variable efficacy, and antivirals, which become ineffective as resistant strains develop. Slow vaccine production and the emergence of new influenza subtypes highlight the need to identify novel targets for development of new antivirals. The influenza viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is highly conserved across multiple influenza subtypes, making this an excellent antiviral target. Nucleoprotein (NP) is an essential component of vRNPs. Our project focuses on defining NP as a novel antiviral target. Our specific aim is to confirm if the small molecule Nucleozin disrupts NP-PB2 interaction. NP amino acids needed for PB2 interaction and vRNP function are the same amino acids that bind to the inhibitor Nucleozin. Therefore we aim to determine if nucleozin inhibits PB2 interaction and vRNP function using sucrose gradients and reconstituted vRNP assays, respectively. To observe if nucleozin inhibits NP-PB2 binding, 293T cells were transfected and nucleozin was added 24 hours post transfection. After protein isolation and ultracentrifugation, fractions were collected and are currently undergoing analysis by Western blot. We also examined the influence of nucleozin on reconstituted vRNP function. Cells were transfected to express reconstituted vRNPs with GFP-MvRNA template. 48 hours post transfection GFP was observed by fluorescent microscopy and total RNA was isolated and subjected to RT-qPCR analysis. Multiple trials confirm that the presence of nucleozin results in decreased GFP-M expression from reconstituted vRNPs. Our project will help define NP as a novel antiviral target, while confirming Nucleozin as a successful inhibitor. Future research will include screening additional small molecules for inhibition of vRNP function.

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Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

The Effect of Nucleozin on Influenza A Nucleoprotein Interactions

94

Influenza A viruses cause seasonal epidemics of respiratory illness and are responsible for health and economic burdens annually. The virus is controlled with annual vaccines, which have variable efficacy, and antivirals, which become ineffective as resistant strains develop. Slow vaccine production and the emergence of new influenza subtypes highlight the need to identify novel targets for development of new antivirals. The influenza viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is highly conserved across multiple influenza subtypes, making this an excellent antiviral target. Nucleoprotein (NP) is an essential component of vRNPs. Our project focuses on defining NP as a novel antiviral target. Our specific aim is to confirm if the small molecule Nucleozin disrupts NP-PB2 interaction. NP amino acids needed for PB2 interaction and vRNP function are the same amino acids that bind to the inhibitor Nucleozin. Therefore we aim to determine if nucleozin inhibits PB2 interaction and vRNP function using sucrose gradients and reconstituted vRNP assays, respectively. To observe if nucleozin inhibits NP-PB2 binding, 293T cells were transfected and nucleozin was added 24 hours post transfection. After protein isolation and ultracentrifugation, fractions were collected and are currently undergoing analysis by Western blot. We also examined the influence of nucleozin on reconstituted vRNP function. Cells were transfected to express reconstituted vRNPs with GFP-MvRNA template. 48 hours post transfection GFP was observed by fluorescent microscopy and total RNA was isolated and subjected to RT-qPCR analysis. Multiple trials confirm that the presence of nucleozin results in decreased GFP-M expression from reconstituted vRNPs. Our project will help define NP as a novel antiviral target, while confirming Nucleozin as a successful inhibitor. Future research will include screening additional small molecules for inhibition of vRNP function.