The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells.

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

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.</p

Toscana virus (TOSV) penetrates host cells by acid-activated membrane fusion.

(A) A549 cells were exposed to TOSV (MOI ∼10) on ice, subjected to various pH values at 37°C for 90 sec to trigger the virus fusion at the plasma membrane, and then incubated for 7 h at 37°C in the presence of 50 mM NH4Cl to prevent viral penetration from endosomes. Infection was quantified by flow cytometry, and the data were normalized to those from samples where the infection was triggered with a buffer at pH 5.0. (B and C) TOSV particles were bound to A549 cells (B) and iPSC-derived neurons (C) at MOIs 1 and 15, respectively, on ice and then rapidly shifted to 37°C to allow virus internalization. 50 mM NH4Cl was added at the indicated times to block further viral penetration. Infected cells were quantified by flow cytometry, and data were normalized to the samples where NH4Cl was added 80 min (B) and 90 min (C) post-warming. (D) R18-TOSV was bound at MOI 10 to A549 cells on ice and rapidly warmed to 37°C. The increase in fluorescence resulted from the dequenching of the lipid dye R18 after virus fusion with cell membranes, as measured by fluorometry (black line). NH4Cl was used to block virus fusion by neutralizing endosomal pH and, thus, to define the fluorescence background due to spontaneous translocation of the R18 dyes between the viral envelope and the neighboring cell membrane (grey line). The red line shows the virus fusion-specific R18 release, i.e., the black line (fusion + free diffusion) minus the grey line (free diffusion). RU, relative unit. (E) TOSV and Semliki Forest virus (SFV) were bound to A549 cells on ice, and samples were shifted to indicated temperatures for 50 min. Infected cells were then incubated at 37°C for 6 h in the presence of NH4Cl and quantified by flow cytometry. Infection was normalized to that in samples incubated throughout at 37°C. (F) TOSV fusion was assessed in A549 cells for efficiency at various temperatures using the assay in A. Data were normalized to those of samples incubated throughout at 37°C. (G) The binding of R18-TOSV (MOI ∼10) to A549 cells was synchronized on ice. Samples were then warmed to 37°C in a fluorometer, and the fluorescence signal was monitored over 10 min. “+pH” indicates when buffers at pH 5.0 or 7.4 were added to trigger virus fusion. Data were normalized to those at the time point 0. The red line shows the virus fusion-specific R18 release at pH ∼5.0. RU, relative unit. (H) R18-TOSV penetration into A549 cells at MOI 10 was recorded in real time for 90 min using the protocol in D, and triton X-100 was added at the end to induce the dequenching of all R18 molecules associated with bound and internalized virions. The data show the ratio between the fluorescence resulting from viral fusion and that associated with all virions in the cells.</p

Toscana virus (TOSV) shows remarkable adaptability to the endosomal environment to penetrate cells.

(A) The binding of R18-TOSV (MOI ∼10) to A549 cells was synchronized on ice. Cells were then rapidly warmed to 37°C, and virus fusion was triggered by adding acidic buffers within a fluorometer while recording the fluorescence signal. The virus fusion-specific R18 release is shown. Data were normalized to those at the time point 0. RU, relative unit. (B) The half-maximal fluorescence intensity (t1/2) was measured in the series of data obtained in A. n > 3. (C) R18-TOSV particles were pretreated at pH 6.0 or 7.4 and then neutralized as described in Fig 8A before being assessed and analyzed as in panel A. The t1/2 of fusion was calculated from n > 7. T-test with Welch’s correction was applied. *, pD) After the synchronization of TOSV binding at MOI 1 on ice, A549 cells were rapidly warmed to 37°C in the presence of NH4Cl (50 mM). NH4Cl was then washed out at the indicated times to allow endosomal acidification and the acid-activated penetration of infectious TOSV particles. Samples were harvested 6 h later, and infection was analyzed by flow cytometry. Values were normalized to those from samples for which NH4Cl was removed at t0.</p

Toscana virus (TOSV) entry into iPSC-derived human neurons depends on endosomal acidification.

(A to C) iPSC-derived neurons (A) and A549 cells (B and C) were pretreated with agents that elevate endosomal pH at the indicated concentrations and were infected with TOSV at MOI 10 for 8 h and MOI 2 for 6 h, respectively, in the continuous presence of ammonium chloride (NH4Cl), chloroquine, bafilomycin A1, and concanamycin B. Infection was analyzed by flow cytometry, and data were normalized to those of control samples without inhibitor treatment.</p

Entry of Toscana virus (TOSV) into cells.

The time and pH scale on the right side illustrates the typical acidity and timing of cellular cargoes to reach the respective endosomal compartments. +/++ indicate the amount of Rab7a and LAMP-1.</p