Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.
We acknowledge Elodie Chatre and the Imaging Platform Platim, SFR Biosciences, Lyon, as well as Vibor Laketa and the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research (CIID) Heidelberg. The sand fly cell lines were supplied by the Tick Cell Biobank at the University of Liverpool. F.K.M.S. acknowledges support from the Scientific Service Units (SSUs) of ISTA through resources provided by the Electron Microscopy Facility (EMF). This work was supported by CellNetworks Research Group funds and Deutsche Forschungsgemeinschaft (DFG) funding (LO-2338/3-1) and the Agence Nationale de la Recherche (ANR) funding (grant numbers ANR-21-CE11-0012 and ANR-22-CE15-0034), all awarded to P.-Y.L. This work was also supported by the LABEX ECOFECT (ANR-11-LABX-0048) of Université de Lyon (UDL), within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the ANR and by the RESPOND program of the UDL (awarded to P.-Y.L) . C.A. was supported by the Chica and Heinz Schaller Research Group funds, NARSAD 2019 award, a Fritz Thyssen Research Grant, and the SFB1158-S02 grant. L.B-S. is supported by a United Kingdom Biotechnology and Biological Sciences Research Council grant (BB/P024270/1) and a Wellcome Trust grant (223743/Z/21/Z). F.K.M.S acknowledges support from the Austrian Science Fund (FWF, P31445). J.K. received a salary from the DFG (LO-2338/3-1) and then from the ANR (ANR-11-LABX-0048). The salary of Z.M.U. was partially covered by the DFG (LO-2338/3-1). S.K. received a salary from the DFG (SFB1129). We are grateful to the Chinese Scholarship Council (CSC; 201904910701), DAAD/ANID (57451854/62180003), the Rufus A. Kellogg fellowship program (Amherst College, Massachusetts, USA) for awarding fellowships to Q.X., J.C., and H.A.A., respectively.
Koch J, Xin Q, Obr M, et al. The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells. PLoS Pathogens. 2023;19(8). doi:10.1371/journal.ppat.1011562
Koch, J., Xin, Q., Obr, M., Schäfer, A., Rolfs, N., Anagho, H. A., … Lozach, P. Y. (2023). The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells. PLoS Pathogens. Public Library of Science. https://doi.org/10.1371/journal.ppat.1011562
Koch, Jana, Qilin Xin, Martin Obr, Alicia Schäfer, Nina Rolfs, Holda A. Anagho, Aiste Kudulyte, et al. “The Phenuivirus Toscana Virus Makes an Atypical Use of Vacuolar Acidity to Enter Host Cells.” PLoS Pathogens. Public Library of Science, 2023. https://doi.org/10.1371/journal.ppat.1011562.
J. Koch et al., “The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells,” PLoS Pathogens, vol. 19, no. 8. Public Library of Science, 2023.
Koch J, Xin Q, Obr M, Schäfer A, Rolfs N, Anagho HA, Kudulyte A, Woltereck L, Kummer S, Campos J, Uckeley ZM, Bell-Sakyi L, Kräusslich HG, Schur FK, Acuna C, Lozach PY. 2023. The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells. PLoS Pathogens. 19(8), e1011562.
Koch, Jana, et al. “The Phenuivirus Toscana Virus Makes an Atypical Use of Vacuolar Acidity to Enter Host Cells.” PLoS Pathogens, vol. 19, no. 8, e1011562, Public Library of Science, 2023, doi:10.1371/journal.ppat.1011562.
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