A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields

Cell Struct Funct. 2022 Jun 25;47(1):43-53. doi: 10.1247/csf.21047. Epub 2022 Apr 28.


The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.

Keywords: SARS-CoV-2; pseudovirus; severe acute respiratory syndrome coronavirus (SARS-CoV); spike protein; vesicular stomatitis virus (VSV).

MeSH terms

  • SARS-CoV-2
  • Spike Glycoprotein, Coronavirus* / biosynthesis
  • Vesicular stomatitis Indiana virus* / metabolism


  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2

Grants and funding

This work was supported in part by Grants-in-Aid for Transformative Research Areas (A) (#20H05872) and Scientific Research on Innovative Areas (#15H01248, #26115701, #19H05411, #19H04823, and #21H00413) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research (B) (#21H00413) from the Japan Society for the Promotion of Science, Research Program on Emerging and Re-emerging Infectious Diseases (JP20fk0108401) and AMED-CREST (JP20fk0108401) from the Japan Agency for Medical Research and Development, as well as by COVID-19 Drug and Vaccine Development Donation and the Photo-excitonix Project in Hokkaido University.