Event Title

Characterization Of Influenza Nucleoprotein Body Domain As Antiviral Target

Presenter Information

Alicia Davis

Presentation Type

Oral Presentation

College

College of Natural Sciences

Major

Biology

Session Number

2

Location

RM 216

Juror Names

Moderator: Dr. Tomasz Owerkowicz

Start Date

5-21-2015 4:00 PM

End Date

5-21-2015 4:20 PM

Abstract

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by viral nucleoprotein (NP) and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins (vRNPs) responsible for RNA synthesis. NP is a critical structural component of the vRNP but also interacts with both viral and host factors to regulate viral RNA expression. NP is conserved among influenza A isolates, making NP interactions compelling antiviral targets. Here we report characterization of NPbd3, an NP mutant encoding 5 amino acid changes within an accessible region of the NP body domain as determined by NP crystal structure. NPbd3 was designed to target interaction between NP and the RdRP. To characterize NPbd3 we first confirmed NPbd3 expression and localization was as WT-NP by both cellular fractionation and Western Blot, and NP-GFP fusions and fluorescence. Although NPbd3 was expressed, localized, and binds nucleic acid as WT-NP, we found NPbd3 was defective in RNA expression in reconstituted vRNPs and cRNPs, as assessed by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). We hypothesize a disruption in NP interaction, likely with the viral polymerase, results in this severe defect. Our current experiments are focused on determining NPbd3-polymerase interaction using co-immunoprecipitation and co-immunofluorescence with a functional Strep tagged PB2, and vRNP formation using blue native gel analysis. Characterization of NP mutants within this conserved and accessible region of the NP body domain will contribute structural information to facilitate studies of antivirals targeting this highly conserved NP body domain

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May 21st, 4:00 PM May 21st, 4:20 PM

Characterization Of Influenza Nucleoprotein Body Domain As Antiviral Target

RM 216

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by viral nucleoprotein (NP) and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins (vRNPs) responsible for RNA synthesis. NP is a critical structural component of the vRNP but also interacts with both viral and host factors to regulate viral RNA expression. NP is conserved among influenza A isolates, making NP interactions compelling antiviral targets. Here we report characterization of NPbd3, an NP mutant encoding 5 amino acid changes within an accessible region of the NP body domain as determined by NP crystal structure. NPbd3 was designed to target interaction between NP and the RdRP. To characterize NPbd3 we first confirmed NPbd3 expression and localization was as WT-NP by both cellular fractionation and Western Blot, and NP-GFP fusions and fluorescence. Although NPbd3 was expressed, localized, and binds nucleic acid as WT-NP, we found NPbd3 was defective in RNA expression in reconstituted vRNPs and cRNPs, as assessed by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). We hypothesize a disruption in NP interaction, likely with the viral polymerase, results in this severe defect. Our current experiments are focused on determining NPbd3-polymerase interaction using co-immunoprecipitation and co-immunofluorescence with a functional Strep tagged PB2, and vRNP formation using blue native gel analysis. Characterization of NP mutants within this conserved and accessible region of the NP body domain will contribute structural information to facilitate studies of antivirals targeting this highly conserved NP body domain