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INRA, Virologie et Immunologie Moléculaires

2 ARTICLES PUBLISHED IN JoVE

Biochemistry

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Javier A. Jaimes^*1,2, Jean K. Millet^*1,3, Monty E. Goldstein^1,4, Gary R. Whittaker¹, Marco R. Straus¹

¹Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ³Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA, ⁴Department of Cell Biology and Molecular Genetics, University of Maryland

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Immunology and Infection

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

Jean K. Millet^1,2, Tiffany Tang³, Lakshmi Nathan³, Javier A. Jaimes⁴, Hung-Lun Hsu^3,5, Susan Daniel³, Gary R. Whittaker¹

¹Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²INRA, Virologie et Immunologie Moléculaires, ³Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, ⁴Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ⁵Horae Gene Therapy Center, University of Massachusetts Medical School

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.