biostudies-literatureCorliss LNational Institute of Allergy and Infectious Diseases Division of Intramural ResearchNIAID NIH HHSNational Institutes of HealthHeersink School of Medicine, University of Alabama at BirminghamNational Institute of General Medical SciencesNIGMS NIH HHS110451https://www.ebi.ac.uk/biostudies/studies/S-EPMC12336820biostudies-literatureUnknown301(8)Orthoflaviviruses are small, enveloped, positive-sense RNA viruses that cause over 500 million infections globally each year for which there are no antiviral treatments. The viral protease is an attractive target for therapeutics due to its critical functions throughout infection. Many studies have reported on the structure, function, and importance of orthoflavivirus proteases; however, the molecular determinants for cleavage of intracellular substrates by orthoflavivirus proteases and how these factors affect viral fitness are unknown. In this study, we used our fluorescent, protease-activity reporter system to investigate the subcellular determinants involved in orthoflavivirus protease cleavage. By modifying our reporter platform, we identified endoplasmic reticulum (ER) subdomain locaThe Journal of biological chemistrySubcellular determinants of orthoflavivirus protease activity.PMC12336820F31-AI183737R35 GM150638R35-GM150638F31 AI183737Petit CMCorliss LLennemann NJfalseSubcellular determinants of orthoflavivirus protease activity.Orthoflaviviruses are small, enveloped, positive-sense RNA viruses that cause over 500 million infections globally each year for which there are no antiviral treatments. The viral protease is an attractive target for therapeutics due to its critical functions throughout infection. Many studies have reported on the structure, function, and importance of orthoflavivirus proteases; however, the molecular determinants for cleavage of intracellular substrates by orthoflavivirus proteases and how these factors affect viral fitness are unknown. In this study, we used our fluorescent, protease-activity reporter system to investigate the subcellular determinants involved in orthoflavivirus protease cleavage. By modifying our reporter platform, we identified endoplasmic reticulum (ER) subdomain loca2025-01-01T00:00:00Z2025 Jul2026-04-13T17:15:07.507Z2026-04-07T13:34:22.21ZS-EPMC123368204061900410.1016/j.jbc.2025.110451