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Updated: Sep 11, 2025
A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Irreversible furin cleavage site exposure renders immature tick-borne flaviviruses fully infectious
Jiří Holoubek1,2,3, Jiří Salát1,2,3, Milos Matkovic4
1Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
Abstract:
Flavivirus assembly is driven by the envelope glycoproteins pre-membrane (prM) and envelope (E) in the neutral pH environment of the endoplasmic reticulum. Newly budded, spiky particles are exported through the Golgi apparatus, where mildly acidic pH induces a major surface rearrangement. The glycoproteins reorganize into (prM/E)\₂ complexes at the surface of smooth particles, with prM trapped at the E dimer interface, thereby exposing a furin cleavage site (FCS) for proteolytic maturation into infectious virions. Here, we show that in the absence of furin, immature tick-borne flavivirus particles-tick-borne encephalitis virus, Langat virus, and Louping ill virus-remain fully infectious and pathogenic in female BALB/c mice, in contrast to mosquito-borne flaviviruses such as Usutu, West Nile, and Zika viruses. We further show that the FCS in tick-borne viruses remains exposed at neutral pH, allowing furin at the surface of target cells to activate viral fusogenicity, while mosquito-borne counterparts require acidic re-exposure. Mutations increasing the dynamic behavior of the E dimer mimic the mosquito-borne phenotype, with retracted FCS at neutral pH and loss of infectivity. Our multidisciplinary approach-combining virological assays, targeted mutagenesis, structural modeling, and molecular dynamics simulations-highlights the role of E dimer dynamics in regulating flavivirus maturation and infectivity.
Insights
Tick-borne flaviviruses are infectious even without furin cleavage, unlike their mosquito-borne counterparts. This difference is due to their envelope glycoproteins maintaining an exposed cleavage site at neutral pH.
Area of Science:
- Virology
- Structural Biology
- Molecular Dynamics
Background:
- Flavivirus assembly involves envelope glycoproteins (prM, E) in the endoplasmic reticulum.
- Maturation into infectious virions requires proteolytic cleavage of prM by furin at acidic pH in the Golgi apparatus.
Purpose of the Study:
- To investigate the infectivity of immature tick-borne flaviviruses in the absence of furin.
- To elucidate the structural basis for differences in maturation and infectivity between tick-borne and mosquito-borne flaviviruses.
Main Methods:
- Virological assays in cell culture and mouse models (BALB/c mice).
- Targeted mutagenesis of envelope glycoproteins.
- Structural modeling and molecular dynamics simulations.
Main Results:
- Immature tick-borne flaviviruses (tick-borne encephalitis virus, Langat virus, Louping ill virus) are infectious and pathogenic without furin cleavage.
- Tick-borne flaviviruses expose their furin cleavage site (FCS) at neutral pH, unlike mosquito-borne flaviviruses (Usutu, West Nile, Zika viruses) which require acidic pH.
- Mutations enhancing E dimer dynamics mimic the mosquito-borne phenotype, leading to retracted FCS and loss of infectivity.
Conclusions:
- Tick-borne flavivirus maturation is not strictly dependent on acidic pH-induced conformational changes for infectivity.
- The dynamic behavior of the E dimer is a key factor regulating flavivirus maturation and infectivity.
- Differences in E dimer dynamics explain the distinct infectivity profiles of tick-borne and mosquito-borne flaviviruses.
