Presentation Title

Generating Influenza A Virus with NP-FLAG epitope tag to facilitate NP interaction studies

Faculty Mentor

Laura L. Newcomb

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

116

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Influenza A virus is responsible for seasonal epidemics and occasional pandemics. Annual vaccination prevents seasonal spread, but antivirals are the best line of defense for an emerging pandemic. However, antivirals become ineffective with use as resistance emerges, highlighting the need to identify new antiviral targets. Influenza A viral ribonucleoprotein (vRNP) complexes are responsible for viral RNA synthesis. Nucleoprotein (NP) is an essential component of the vRNP complex. NP is relatively consistent in all influenza A lineages, making NP a great antiviral target. Our aim was to create a virus with a 7-amino acid epitope tag (NP-FLAG) attached to NP. This will provide a tool to the influenza community to examine NP interactions throughout the virus life cycle. Our initial attempts indicated that the NP viral RNA genome packaging signal was disrupted by addition of the C-terminus FLAG tag. To resolve this issue, we constructed a plasmid to express the NP-FLAG vRNA segment with a repeat of NP coding sequence without disruption, to provide the vRNA segment packaging signal. 293t cells were transfected with the 12 plasmids needed to generate virus. After 48 hrs, 293t cells were overlaid onto MDCK cells to amplify virus. Media samples were collected at different time points and hemagglutination assay (HA) was performed. Our results reveal generation of virion particles with WT-NP and NP-FLAG, while our negative control with no NP segment did not. The NP-FLAG media was injected into specific pathogen free eggs to amplify virus for further analysis. Recovery yielded a significantly higher HA titer, confirming virus amplification. NP-FLAG protein expression will be verified through western blot, and NP-FLAG segment by RT-PCR using FLAG specific primers. In addition, all segments will be sequenced. Overall, our research will provide a tool to facilitate research of NP interactions and identify new antiviral targets.

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

Generating Influenza A Virus with NP-FLAG epitope tag to facilitate NP interaction studies

116

Influenza A virus is responsible for seasonal epidemics and occasional pandemics. Annual vaccination prevents seasonal spread, but antivirals are the best line of defense for an emerging pandemic. However, antivirals become ineffective with use as resistance emerges, highlighting the need to identify new antiviral targets. Influenza A viral ribonucleoprotein (vRNP) complexes are responsible for viral RNA synthesis. Nucleoprotein (NP) is an essential component of the vRNP complex. NP is relatively consistent in all influenza A lineages, making NP a great antiviral target. Our aim was to create a virus with a 7-amino acid epitope tag (NP-FLAG) attached to NP. This will provide a tool to the influenza community to examine NP interactions throughout the virus life cycle. Our initial attempts indicated that the NP viral RNA genome packaging signal was disrupted by addition of the C-terminus FLAG tag. To resolve this issue, we constructed a plasmid to express the NP-FLAG vRNA segment with a repeat of NP coding sequence without disruption, to provide the vRNA segment packaging signal. 293t cells were transfected with the 12 plasmids needed to generate virus. After 48 hrs, 293t cells were overlaid onto MDCK cells to amplify virus. Media samples were collected at different time points and hemagglutination assay (HA) was performed. Our results reveal generation of virion particles with WT-NP and NP-FLAG, while our negative control with no NP segment did not. The NP-FLAG media was injected into specific pathogen free eggs to amplify virus for further analysis. Recovery yielded a significantly higher HA titer, confirming virus amplification. NP-FLAG protein expression will be verified through western blot, and NP-FLAG segment by RT-PCR using FLAG specific primers. In addition, all segments will be sequenced. Overall, our research will provide a tool to facilitate research of NP interactions and identify new antiviral targets.