Pseudotyped vesicular stomatitis virus for analysis of virus entry mediated by SARS coronavirus spike proteins.
Identifieur interne : 001A15 ( PubMed/Corpus ); précédent : 001A14; suivant : 001A16Pseudotyped vesicular stomatitis virus for analysis of virus entry mediated by SARS coronavirus spike proteins.
Auteurs : Shuetsu Fukushi ; Rie Watanabe ; Fumihiro TaguchiSource :
- Methods in molecular biology (Clifton, N.J.) [ 1064-3745 ] ; 2008.
English descriptors
- KwdEn :
- Animals, Cell Line, Chlorocebus aethiops, Green Fluorescent Proteins (genetics), Green Fluorescent Proteins (metabolism), Humans, Membrane Glycoproteins (metabolism), Microscopy, Fluorescence, Models, Biological, SARS Virus (metabolism), SARS Virus (physiology), Spike Glycoprotein, Coronavirus, Vero Cells, Vesicular stomatitis Indiana virus (genetics), Vesicular stomatitis Indiana virus (metabolism), Vesicular stomatitis Indiana virus (physiology), Viral Envelope Proteins (metabolism), Virus Internalization.
- MESH :
- chemical , genetics : Green Fluorescent Proteins.
- chemical , metabolism : Green Fluorescent Proteins, Membrane Glycoproteins, Viral Envelope Proteins.
- genetics : Vesicular stomatitis Indiana virus.
- metabolism : SARS Virus, Vesicular stomatitis Indiana virus.
- physiology : SARS Virus, Vesicular stomatitis Indiana virus.
- Animals, Cell Line, Chlorocebus aethiops, Humans, Microscopy, Fluorescence, Models, Biological, Spike Glycoprotein, Coronavirus, Vero Cells, Virus Internalization.
Abstract
Severe acute respiratory syndrome (SARS) coronavirus (CoV) contains a spike (S) protein that binds to a receptor molecule (angiotensin-converting enzyme 2; ACE2), induces membrane fusion, and serves as a neutralizing epitope. To study the functions of the S protein, we describe here the generation of SARS-CoV S protein-bearing vesicular stomatitis virus (VSV) pseudotype using a VSVdeltaG*/GFP system in which the G gene is replaced by the green fluorescent protein (GFP) gene (VSV-SARS-CoV-St19/GFP). Partial deletion of the cytoplasmic domain of SARS-CoV S protein (SARS-CoV-St19) allowed efficient incorporation into the VSV particle that enabled the generation of a high titer of pseudotype virus. Neutralization assay with anti-SARS-CoV antibody revealed that VSV-SARS-St19/GFP pseudotype infection is mediated by SARS-CoV S protein. The VSVdeltaaG*/SEAP system, which secretes alkaline phosphatase instead of GFP, was also generated as a VSV pseudotype having SARS-CoV S protein (VSV-SARS-CoV-St19/SEAP). This system enabled high-throughput analysis of SARS-CoV S protein-mediated cell entry by measuring alkaline phosphatase activity. Thus, VSV pseudotyped with SARS-CoV S protein is useful for developing a rapid detection system for neutralizing antibody specific for SARS-CoV infection as well as studying the S-mediated cell entry of SARS-CoV.
DOI: 10.1007/978-1-59745-181-9_23
PubMed: 19057867
Links to Exploration step
pubmed:19057867Le document en format XML
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<author><name sortKey="Fukushi, Shuetsu" sort="Fukushi, Shuetsu" uniqKey="Fukushi S" first="Shuetsu" last="Fukushi">Shuetsu Fukushi</name>
<affiliation><nlm:affiliation>Department of Virology I, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.</nlm:affiliation>
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<author><name sortKey="Watanabe, Rie" sort="Watanabe, Rie" uniqKey="Watanabe R" first="Rie" last="Watanabe">Rie Watanabe</name>
</author>
<author><name sortKey="Taguchi, Fumihiro" sort="Taguchi, Fumihiro" uniqKey="Taguchi F" first="Fumihiro" last="Taguchi">Fumihiro Taguchi</name>
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<term>Green Fluorescent Proteins (metabolism)</term>
<term>Humans</term>
<term>Membrane Glycoproteins (metabolism)</term>
<term>Microscopy, Fluorescence</term>
<term>Models, Biological</term>
<term>SARS Virus (metabolism)</term>
<term>SARS Virus (physiology)</term>
<term>Spike Glycoprotein, Coronavirus</term>
<term>Vero Cells</term>
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<term>Viral Envelope Proteins (metabolism)</term>
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<front><div type="abstract" xml:lang="en">Severe acute respiratory syndrome (SARS) coronavirus (CoV) contains a spike (S) protein that binds to a receptor molecule (angiotensin-converting enzyme 2; ACE2), induces membrane fusion, and serves as a neutralizing epitope. To study the functions of the S protein, we describe here the generation of SARS-CoV S protein-bearing vesicular stomatitis virus (VSV) pseudotype using a VSVdeltaG*/GFP system in which the G gene is replaced by the green fluorescent protein (GFP) gene (VSV-SARS-CoV-St19/GFP). Partial deletion of the cytoplasmic domain of SARS-CoV S protein (SARS-CoV-St19) allowed efficient incorporation into the VSV particle that enabled the generation of a high titer of pseudotype virus. Neutralization assay with anti-SARS-CoV antibody revealed that VSV-SARS-St19/GFP pseudotype infection is mediated by SARS-CoV S protein. The VSVdeltaaG*/SEAP system, which secretes alkaline phosphatase instead of GFP, was also generated as a VSV pseudotype having SARS-CoV S protein (VSV-SARS-CoV-St19/SEAP). This system enabled high-throughput analysis of SARS-CoV S protein-mediated cell entry by measuring alkaline phosphatase activity. Thus, VSV pseudotyped with SARS-CoV S protein is useful for developing a rapid detection system for neutralizing antibody specific for SARS-CoV infection as well as studying the S-mediated cell entry of SARS-CoV.</div>
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