The Orthobunyavirus Germiston Enters Host Cells from Late Endosomes

Abstract

With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30-40 min. The virus entered Rab5a+ early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15-25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here we developed accurate, sensitive tools and assays to investigate the penetration process of the OBV Germiston (GERV). Our data emphasize the central role of the late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lay the foundation for the development of antiviral strategies aiming to block OBV entry.

The glycolipid GlcCer is recruited into the viral envelope to promote phenuivirus binding to host cells

“…Our results indicate that GlcCer was also embedded in both TOSV and GERV particles. GERV belongs to the Peribunyaviridae family, which is closely related to the Phenuiviridae family (Windhaber et al, 2022), and similar like phenuiviruses, peribunyaviruses bud into Golgi compartments (Elliott, 2014; Elliott and Brennan, 2014; Léger and Lozach, 2015). In contrast, SFV and EBOVLPs assemble at the plasma membrane, and our analysis showed no significant level of GlcCer level in their respective viral envelope.…”

“…UUKV strain 23 was obtained from plasmids and amplified in BHK-21 cells (Mazelier et al 2016). The prototype strains of SFV, TOSV ISS, and GERV have been described previously (Pettersson and Kääriäinen, 1973; Helenius et al, 1980; Giorgi et al, 1991; Overby et al, 2006; Windhaber et al, 2022). All viruses were produced in BHK-21 cells and purified and titrated according to standard procedures (Meier et al, 2014; Léger et al, 2016; Hoffmann et al, 2018; Windhaber et al, 2022).…”

“…The prototype strains of SFV, TOSV ISS, and GERV have been described previously (Pettersson and Kääriäinen, 1973; Helenius et al, 1980; Giorgi et al, 1991; Overby et al, 2006; Windhaber et al, 2022). All viruses were produced in BHK-21 cells and purified and titrated according to standard procedures (Meier et al, 2014; Léger et al, 2016; Hoffmann et al, 2018; Windhaber et al, 2022). Fluorescent labeling was performed with one molecule of viral glycoproteins per three molecules of ATTO488 dye (Atto-Tec) in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) following a protocol established by our group (Hoffmann et al, 2018).…”

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

“…Bafilomycin A1 (BioViotica), colcemid (Cayman Chemical), concanamycin B (BioViotica), MG-132 (Selleck Chemicals), nocodazole (Merck) and taxol (Merck) were all dissolved in 100% dimethyl sulfoxide (DMSO) to prepare stock solutions at 100 µM, 10 mM, 50 µM, 40 mM, 20 mM and 10 mM, respectively. Stock solutions of all drugs were diluted in DMEM at the indicated doses (Figures 1 and 4), which are known not to cause cytotoxicity ( 23, 34 ). Both dH 2 O and DMSO were included as solvent controls.…”

Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry

“…However, the low resolution of our subtomogram average (STA) and the lack of atomic models for HAZV GPs prevented a detailed structural understanding of the effect of K + on the GPs. Of note, it has recently been shown that disruption of the cellular K + gradient using KCl or the K + ionophore valinomycin inhibits infection by the peribunyaviruses La Crosse virus (LACV), keystone virus and Germiston virus; and of the phenuivirus RVFV (14,15), suggesting that K + is broadly required during bunyavirus entry.…”