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Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein.
J Virol. 2020 10 14; 94(21)JV

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.

Authors+Show Affiliations

Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA marcjohnson@missouri.edu.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri, USA.Department of Anatomical and Clinical Pathology, MU Health Care, Columbia, Missouri, USA.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

32788194

Citation

Johnson, Marc C., et al. "Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein." Journal of Virology, vol. 94, no. 21, 2020.
Johnson MC, Lyddon TD, Suarez R, et al. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. J Virol. 2020;94(21).
Johnson, M. C., Lyddon, T. D., Suarez, R., Salcedo, B., LePique, M., Graham, M., Ricana, C., Robinson, C., & Ritter, D. G. (2020). Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. Journal of Virology, 94(21). https://doi.org/10.1128/JVI.01062-20
Johnson MC, et al. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. J Virol. 2020 10 14;94(21) PubMed PMID: 32788194.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. AU - Johnson,Marc C, AU - Lyddon,Terri D, AU - Suarez,Reinier, AU - Salcedo,Braxton, AU - LePique,Mary, AU - Graham,Maddie, AU - Ricana,Clifton, AU - Robinson,Carolyn, AU - Ritter,Detlef G, Y1 - 2020/10/14/ PY - 2020/05/27/received PY - 2020/08/06/accepted PY - 2020/8/14/pubmed PY - 2020/10/28/medline PY - 2020/8/14/entrez KW - COVID-19 KW - SARS-COV-2 KW - glycoprotein KW - lentiviral vector KW - neutralization assay KW - pseudotypes JF - Journal of virology JO - J Virol VL - 94 IS - 21 N2 - The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/32788194/Optimized_Pseudotyping_Conditions_for_the_SARS_COV_2_Spike_Glycoprotein_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=32788194 DB - PRIME DP - Unbound Medicine ER -