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.

Department of Clinical Microbiology Laboratory for Molecular Infection Medicine Sweden Umeå University Umeå Sweden

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Infection Biology and Microbiomes Institute of Infection Ecological and Veterinary Sciences University of Liverpool Liverpool United Kingdom

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Prague Czech Republic

Institut Pasteur Université de Paris Cité CNRS UMR 3569 Structural Virology Unit Paris France

Institut Pasteur Université Paris Cité CNRS UMR 3528 Computational Structural Biology Unit Paris France

Institute for Research in Biomedicine Università della Svizzera Italiana Bellinzona Switzerland

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Laboratory of Emerging Viral Diseases Veterinary Research Institute Brno Czech Republic

Swiss Institute of Bioinformatics Lausanne Switzerland

Zobrazit více v PubMed

Pierson, T. C. & Diamond, M. S. The continued threat of emerging flaviviruses. PubMed PMC

Gould, E. & Solomon, T. Pathogenic flaviviruses. PubMed

Chiffi, G., Grandgirard, D., Leib, S. L., Chrdle, A. & Ruzek, D. Tick-borne encephalitis: a comprehensive review of the epidemiology, virology, and clinical picture. PubMed

Campbell, G. L., Marfin, A. A., Lanciotti, R. S. & Gubler, D. J. West Nile virus. PubMed

Solomon, T. & Vaughn, D. W. Pathogenesis and clinical features of Japanese encephalitis and West Nile virus infections. PubMed

Petersen, L. R., Jamieson, D. J., Powers, A. M. & Honein, M. A. Zika Virus. PubMed

Mackenzie, J. S., Gubler, D. J. & Petersen, L. R. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. PubMed

Gould, E., Pettersson, J., Higgs, S., Charrel, R. & de Lamballerie, X. Emerging arboviruses: why today?. PubMed PMC

Ruzek, D. et al. Tick-borne encephalitis in Europe and Russia: review of pathogenesis, clinical features, therapy, and vaccines. PubMed

Mansbridge, C. T. et al. Autochthonous tick-borne encephalitis in the United Kingdom: a second probable human case and local eco-epidemiological findings. PubMed

Fares, W. et al. Tick-borne encephalitis virus in Ixodes ricinus (Acari: Ixodidae) ticks, Tunisia. PubMed

Lindenbach, B. D. & Rice, C. M. Molecular biology of flaviviruses. PubMed

Barnard, T. R., Abram, Q. H., Lin, Q. F., Wang, A. B. & Sagan, S. M. Molecular Determinants of Flavivirus Virion Assembly. PubMed

Stadler, K., Allison, S. L., Schalich, J. & Heinz, F. X. Proteolytic activation of tick-borne encephalitis virus by furin. PubMed PMC

Plevka, P., Battisti, A. J., Sheng, J. & Rossmann, M. G. Mechanism for maturation-related reorganization of flavivirus glycoproteins. PubMed PMC

Carro, S. D. & Cherry, S. Beyond the surface: endocytosis of mosquito-borne flaviviruses. PubMed PMC

Hackett, B. A. & Cherry, S. Flavivirus internalization is regulated by a size-dependent endocytic pathway. PubMed PMC

Dahmane, S. et al. Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation. Preprint at

Miorin, L. et al. Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA. PubMed PMC

Yau, W.-L. et al. Model system for the formation of tick-borne encephalitis virus replication compartments without viral RNA replication. PubMed PMC

Yu, I.-M. et al. Association of the pr peptides with dengue virus at acidic pH blocks membrane fusion. PubMed PMC

Vaney, M.-C. et al. Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery. PubMed PMC

Nicholls, C. M. R., Sevvana, M. & Kuhn, R. J. Structure-guided paradigm shifts in flavivirus assembly and maturation mechanisms. PubMed PMC

Renner, M. et al. Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins. PubMed PMC

Zheng, A., Yuan, F., Kleinfelter, L. M. & Kielian, M. A toggle switch controls the low pH-triggered rearrangement and maturation of the dengue virus envelope proteins. PubMed PMC

Mukherjee, S. et al. Enhancing dengue virus maturation using a stable furin over-expressing cell line. PubMed PMC

Yu, I. M. et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. PubMed

Zhang, Y., Kaufmann, B. R., Chipman, P. R., Kuhn, R. J. & Rossmann, M. G. Structure of immature West Nile virus. PubMed PMC

Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. PubMed

Hardy, J. M. et al. A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses. PubMed PMC

Kuhn, R. J. et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. PubMed PMC

Zhang, X. et al. Cryo-EM structure of the mature dengue virus at 3.5-A resolution. PubMed PMC

Luca, V. C., AbiMansour, J., Nelson, C. A. & Fremont, D. H. Crystal structure of the Japanese encephalitis virus envelope protein. PubMed PMC

Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. A ligand-binding pocket in the dengue virus envelope glycoprotein. PubMed PMC

Crampon, E. et al. New insight into flavivirus maturation from structure/function studies of the yellow fever virus envelope protein complex. PubMed PMC

Anastasina, M. et al. The structure of immature tick-borne encephalitis virus. PubMed PMC

Pierson, T. C. & Diamond, M. S. Degrees of maturity: the complex structure and biology of flaviviruses. PubMed PMC

Stiasny, K., Medits, I., Roßbacher, L. & Heinz, F. X. Impact of structural dynamics on biological functions of flaviviruses. PubMed PMC

Zybert, I. A., van der Ende-Metselaar, H., Wilschut, J. & Smit, J. M. Functional importance of dengue virus maturation: infectious properties of immature virions. PubMed

Braun, E. & Sauter, D. Furin-mediated protein processing in infectious diseases and cancer. PubMed PMC

Pichkur, E. B. et al. The structure of inactivated mature tick-borne encephalitis virus at 3.0 A resolution. PubMed PMC

Pulkkinen, L. I. A. et al. Molecular Organisation of Tick-Borne Encephalitis Virus. PubMed PMC

Medits, I. et al. Extensive flavivirus E trimer breathing accompanies stem zippering of the post-fusion hairpin. PubMed PMC

Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. PubMed PMC

Zondlo, N. J. Aromatic-proline interactions: electronically tunable CH/pi interactions. PubMed PMC

Prasad, V. M. et al. Structure of the immature Zika virus at 9 Å resolution. PubMed PMC

Zhang, Y. Structures of immature flavivirus particles. PubMed PMC

Rodenhuis-Zybert, I. A. et al. Immature dengue virus: a veiled pathogen?. PubMed PMC

Dejnirattisai, W. et al. Cross-reacting antibodies enhance dengue virus infection in humans. PubMed PMC

Rodenhuis-Zybert, I. A., Wilschut, J. & Smit, J. M. Partial maturation: an immune-evasion strategy of dengue virus?. PubMed

Molloy, S. S., Anderson, E. D., Jean, F. & Thomas, G. Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. PubMed

Newton, N. D. et al. The structure of an infectious immature flavivirus redefines viral architecture and maturation. PubMed PMC

Johnson, A., Dodes Traian, M., Walsh, R. M. Jr., Jenni, S. & Harrison, S. C. Octahedral small virus-like particles of dengue virus type 2. PubMed PMC

Pontremoli, C., Forni, D., Clerici, M., Cagliani, R. & Sironi, M. Alternation between taxonomically divergent hosts is not the major determinant of flavivirus evolution. PubMed PMC

Hönig, V. et al. Multiple Lineages of Usutu Virus ( PubMed PMC

Rudolf, I. et al. West Nile virus lineage 2 isolated from PubMed

Bell-Sakyi, L., Zweygarth, E., Blouin, E. F., Gould, E. A. & Jongejan, F. Tick cell lines: tools for tick and tick-borne disease research. PubMed

Růzek, D., Bell-Sakyi, L., Kopecký, J. & Grubhoffer, L. Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks. PubMed

Takahashi, S. et al. A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. PubMed

De Madrid, A. T. & Porterfield, J. S. A simple micro-culture method for the study of group B arboviruses. PubMed PMC

Haviernik, J. et al. Development and characterization of recombinant tick-borne encephalitis virus expressing mCherry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays. PubMed

Niedrig, M. et al. Monoclonal antibodies directed against tick-borne encephalitisvirus with neutralizing activity in vivo. PubMed

Kubinski, M. et al. Immunity to tick-borne encephalitis virus NS3 protein induced with a recombinant modified vaccinia virus ankara fails to afford mice protection against TBEV infection. PubMed PMC

Starodubova, E. et al. Activation of early proinflammatory responses by TBEV NS1 varies between the strains of various subtypes. PubMed PMC

Finkensieper, J. et al. Low-energy electron irradiation of tick-borne encephalitis virus provides a protective inactivated vaccine. PubMed PMC

Eyer, L. et al. Escape of tick-borne flavivirus from 2’-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness. PubMed PMC

Eyer, L. et al. An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor galidesivir (BCX4430) and also attenuates the virus for mice. PubMed PMC

Salát, J. et al. Development and testing of a new tick-borne encephalitis virus vaccine candidate for veterinary use. PubMed

Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. PubMed

Izidoro, M. A. et al. A study of human furin specificity using synthetic peptides derived from natural substrates, and effects of potassium ions. PubMed PMC

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. PubMed PMC

Füzik, T. et al. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. PubMed PMC

Khare, B., Klose, T., Fang, Q., Rossmann, M. G. & Kuhn, R. J. Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry. PubMed PMC

Aubry, F. et al. Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons. PubMed PMC

Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. PubMed

Shen, M. Y. & Sali, A. Statistical potential for assessment and prediction of protein structures. PubMed PMC

Lomize, A. L., Todd, S. C. & Pogozheva, I. D. Spatial arrangement of proteins in planar and curved membranes by PPM 3.0. PubMed PMC

Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. PubMed

Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. PubMed PMC

MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. PubMed

Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. PubMed

Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method.

Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations.

Essmann, U. et al. A smooth particle mesh Ewald method.

Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.

Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. PubMed PMC

Bonomi, M. et al. Promoting transparency and reproducibility in enhanced molecular simulations. PubMed