Presentation Title

Method for VSV-G and Spike Viral Pseudotyping

Presenter Information

Nathan BooherFollow

Advisor Information

Dr. Paul W Denton

Location

MBSC Ballroom - Poster #108 - U

Presentation Type

Poster

Start Date

4-3-2022 12:30 PM

End Date

4-3-2022 1:45 PM

Abstract

Method for VSV-G and Spike Viral Pseudotyping

Nathan Booher, Kelcy Swope, Paul W. Denton Ph.D. Department of Biology, University of Nebraska Omaha

Introduction

We are using a virus pseudotyping system to screen drugs that interfere with SARS-CoV-2 entry. To date, we have been focused on establishing the system in our laboratory.

Methods

We use a triple transfection approach in HEK-293T cells for pseudotyped virion production. Plasmid 1: The backbone virus for virion production is a Moloney Murine Leukemia Virus (MoMLV). Plasmid 2: This is a psi-element containing plasmid that encodes an EGFP reporter gene. Plasmid 3: This is a plasmid encoding the SARS-CoV-2 spike protein (variants will be introduced). Control Plasmid 3 encodes the envelope for Vesicular Stomatitis Virus G (VSVG). Viral particles are transduced onto VERO E6 cells which express the receptor for spike-mediated entry – ACE2. Transduction efficiency (proxy for viral entry) is quantitated via flow cytometry. Transfection was also monitored via fluorescence microscopy.

Results, Discussion

Initial results yielded a transduction efficiency of 3.4% with VSVG and lower with spike. This is not as high as we believe is possible and the result reveals that our pseudotyping method can be enhanced. Next steps include performing transductions in the presence of transduction enhancing agents (e.g polybrene). Other next steps include modulating plasmid rations during transfection to enhance virion production with maximal viral envelope incorporation. Once this system is functioning, we will work with our collaborators at UNMC to screen drugs with potential for interfering with SARS-CoV-2 entry into target cells.

This document is currently not available here.

COinS
 
Mar 4th, 12:30 PM Mar 4th, 1:45 PM

Method for VSV-G and Spike Viral Pseudotyping

MBSC Ballroom - Poster #108 - U

Method for VSV-G and Spike Viral Pseudotyping

Nathan Booher, Kelcy Swope, Paul W. Denton Ph.D. Department of Biology, University of Nebraska Omaha

Introduction

We are using a virus pseudotyping system to screen drugs that interfere with SARS-CoV-2 entry. To date, we have been focused on establishing the system in our laboratory.

Methods

We use a triple transfection approach in HEK-293T cells for pseudotyped virion production. Plasmid 1: The backbone virus for virion production is a Moloney Murine Leukemia Virus (MoMLV). Plasmid 2: This is a psi-element containing plasmid that encodes an EGFP reporter gene. Plasmid 3: This is a plasmid encoding the SARS-CoV-2 spike protein (variants will be introduced). Control Plasmid 3 encodes the envelope for Vesicular Stomatitis Virus G (VSVG). Viral particles are transduced onto VERO E6 cells which express the receptor for spike-mediated entry – ACE2. Transduction efficiency (proxy for viral entry) is quantitated via flow cytometry. Transfection was also monitored via fluorescence microscopy.

Results, Discussion

Initial results yielded a transduction efficiency of 3.4% with VSVG and lower with spike. This is not as high as we believe is possible and the result reveals that our pseudotyping method can be enhanced. Next steps include performing transductions in the presence of transduction enhancing agents (e.g polybrene). Other next steps include modulating plasmid rations during transfection to enhance virion production with maximal viral envelope incorporation. Once this system is functioning, we will work with our collaborators at UNMC to screen drugs with potential for interfering with SARS-CoV-2 entry into target cells.