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

Abstract

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 but inefficient, 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.

Illuminating bunyavirus entry into host cells with fluorescence

“…Infectious titers typically reach 10 7 infectious particles per milliliter in cell supernatants, and higher numbers are achieved when considering both infectious and noninfectious particles (Hoffmann et al, 2018). The integrity of bunyaviral particles is largely preserved after labeling, although some isolates may require adjustments to reach a balance among the dye amount, virion fluorescence intensity, and infectivity (Koch et al, 2023; Lozach, Kuhbacher, et al, 2011; Windhaber et al, 2022). The fluorescent labeling of viral particles with small organic dyes has proven successful in analyzing each step of the entry program of several bunyaviruses, as described below.…”

“…Since then, several other bunyaviruses have been successfully labeled with thiol‐ and amine‐reactive fluorescent dyes to examine the entry process in fixed and live cells (Table 1). Confocal microscopy, flow cytometry, and fluorometry were used to monitor the binding of the phenuivirus Toscana virus (TOSV), the peribunyavirus Germiston virus (GERV), and the arenavirus Junín virus to the plasma membrane (Chou et al, 2016; Koch et al, 2023; Windhaber et al, 2022). Real‐time imaging using total internal reflection (TIRF) microscopy revealed the motion of UUKV on the cell surface upon virus attachment and elucidated how phenuiviruses exploit the receptors DC‐SIGN and L‐SIGN, two human C‐type lectins, to enter host cells (Leger et al, 2016; Lozach, Kuhbacher, et al, 2011).…”

“…Once attached to the cell surface, bunyaviruses undergo endocytosis. By using the BODIPY TR, AF, and Atto dyes, some bunyaviruses were directly observed in endosomes, and their journey through the endocytic machinery was examined using TIRF and confocal microscopy in both fixed and live cells (Chou et al, 2016; Koch et al, 2023; Lozach et al, 2010; Windhaber et al, 2022). In complementary approaches, differentiation between virions on the cell surface and those internalized within endosomes was achieved by exploiting the photophysical and biochemical properties of the BODIPY TR and AF/Atto dyes.…”

“…Consequently, after treatment, only internalized virions remain fluorescent and can be seen by microscopy or quantified by flow cytometry or fluorimetry (Lozach et al, 2010; Meier et al, 2014). With this method, bunyavirus binding to cells was found to be relatively inefficient but rapid, with the internalization process lasting 10–25 min depending on the viral isolate and family and the target cells (Koch et al, 2023; Lozach, Kuhbacher, et al, 2011; Meier et al, 2014; Windhaber et al, 2022).…”

“…Although the subsequent release of the dye into the target membranes corresponds to the hemifused mixture of outer leaflets rather than the strict formation of a fusion pore, it serves as a good correlate for fusion. This approach has been successfully employed to measure the fusion of UUKV, GERV, and TOSV directly in live cells (Hoffmann et al, 2018; Koch et al, 2023; Meier et al, 2014; Windhaber et al, 2022) (Table 1). These viruses typically require 15–40 min from the moment they bind to the cell surface to enter cells and fuse in late endosomal compartments.…”