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. 2008;454:331-8.
doi: 10.1007/978-1-59745-181-9_23.

Pseudotyped Vesicular Stomatitis Virus for Analysis of Virus Entry Mediated by SARS Coronavirus Spike Proteins

Free PMC article

Pseudotyped Vesicular Stomatitis Virus for Analysis of Virus Entry Mediated by SARS Coronavirus Spike Proteins

Shuetsu Fukushi et al. Methods Mol Biol. .
Free PMC article


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.


Fig. 1
Fig. 1
Schematic illustration of the production and infection of VSV pseudotype bearing SARS-CoV S protein: (1) Transfection of pKS/SARS-St19. It provides the S protein of SARS-CoV and cells express the S protein on the cell surface. (2) Infection of VSV∆G*/GFP-G or VSV∆G*/SEAP-G. These viruses possess the genome containing the reporter gene instead of the VSV-G gene. (3) Virus replication and translation. All viral components except the G protein will be supplied by these viruses. (4) Virus assembly, budding, and pseudotyping. Translated viral proteins are assembled and viral particles bud from the plasma membrane. Since the S protein, which was provided by the expression plasmid, is expressed on the cell surface, a virus can incorporate it into the virus particle. (5) VSV-SARS-St19/GFP and VSV-SARS-St19/SEAP. Pseudotyped VSV possessing SARS-S protein is released into culture supernatant. (6) Infection of the pseudotyped VSV. These viral particles can infect cells expressing the receptor for SARS-CoV, ACE2. (7) Estimation of the infectivity of the viruses. The infection of the pseudotyped viruses is estimated by the expression level of the reporter protein. **Progeny viruses are produced by the infection of VSV-SARS-St19/GFP or VSV-SARS-St19/SEAP. However, these viruses do not have infectivity because they have no glycoprotein (virus with the thin lines).
Fig. 2.
Fig. 2.
Detection of VSV-SARS-St19/GFP infection. The VSV-SARS-St19/GFP was inoculated to Vero E6 cells. GFP expression was examined under a fluorescent microscope.
Fig. 3.
Fig. 3.
Neutralization of infection of VSV-SARS-St19/GFP. The VSV-SARS-St19/GFP mixed with serially diluted rabbit anti-SARS-CoV was inoculated into Vero E6 cells. Rabbit anti-SARS-CoV-N peptide was used as negative control serum. The number of GFP-positive cells in the absence of serum was set as 100%.

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