ESCRT machinery components are required for Orthobunyavirus particle production in Golgi compartments

Abstract

Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.

Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

“…For that, we efficiently standardized an RNA PrimeFlow™ protocol with RNA probes to identify the OROV genome and antigenome in infected cells. We could also evaluate OROV formation of viral factories, where the RNA probes colocalized with the TNG46 protein, corroborating with a previous study [35], demonstrating the efficiency of the assay. This is an important approach to consider since the options of anti-OROV antibodies in the market are very restricted.…”

“…In addition, OROV genetic material and viral antigens colocalizing with trans-Golgi network marker TNG46 (Supplemental Figure S2), which is in agreement with previously published studies showing that the Golgi complex is the main assembly site for OROV [35]. Thus, this innovative technique based on RNA hybridization was considered adequate to differentiate cells in which OROV is only internalized from those in which OROV is able to replicate.…”

The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity

“…Partial deletion of NSm first TMD and the cytosolic loop resulted in lower density of tubes, decreased virus yields and intracellular accumulation of immature viral intermediates. Similarly, Oropouche virus (OROV) proteins accumulate in vesicular structures derived from enlarged Golgi cisternae and recruits ESCRT machinery elements to these viral factories derived from the Golgi cisternae [37]. Likewise, the regulation of trafficking rates and GP N-glycosylation by NSm could be an important mechanism evolved by CCHFV to overcome Golgi retention after sufficient accumulation of virus components required for particle formation has occurred and to promote the release of progeny virus.…”

PI4KB on Inclusion Bodies Formed by ER Membrane Remodeling Facilitates Replication of Human Parainfluenza Virus Type 3

“…Bunyamwera virus infection induces fragmented Golgi stacks, where assembly and maturation take place (Novoa et al, 2005b;Salanueva et al, 2003). Oropouche virus attracts endosomal sorting complex required for transport (ESCRT) machinery components to modify Golgi cisternae, forming viral factory units (Barbosa et al, 2018). However, compared with positive-strand RNA viruses, the IB formation mechanisms of many negative-strand RNA viruses remain unexplored.…”