Pseudotyped vesicular stomatitis virus for analysis of virus entry mediated by SARS coronavirus spike proteins.
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.
Fukushi
Shuetsu
S
Department of Virology I, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.
Watanabe
Rie
R
Taguchi
Fumihiro
F
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism* / Q000502:physiology; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014759:Viral Envelope Proteins / Q000378:metabolism*; D053586:Virus Internalization
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D049452:Green Fluorescent Proteins