AbstractOropouche virus (OROV; Genus: Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. To facilitate studies of OROV budding and assembly, we developed a system for producing OROV virus-like particles (VLPs). Using this system we show that the OROV surface glycoproteins Gn and Gc self-assemble to form VLPs independently of the non-structural protein NSm. Mature OROV Gn has two trans-membrane domains that are crucial for glycoprotein translocation to the Golgi complex and VLP production. Inhibition of Golgi function using the drugs brefeldin A and monensin inhibit VLP secretion, with monensin treatment leading to an increase in co-localisation of OROV glycoproteins with the cis-Golgi marker protein GM130. Infection studies have previously shown that the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. We demonstrate that a dominant negative form of the ESCRT-associated ATPase VPS4 significantly reduces Gn secretion in our VLP assay. Proteasome inhibition using the drug MG132 also disrupts VLPs secretion, suggesting that ubiquitylation promotes ESCRT-mediated VLP release. Additionally, we observe co-localisation between OROV glycoproteins and a specific subset of fluorescently-tagged ESCRT-III components, providing the first insights into which ESCRT-III components are required for OROV assembly. Our in vitro assay for OROV VLP production has allowed us to define molecular interactions that promote OROV release and will facilitate future studies of orthobunyavirus assembly.ImportanceOropouche virus is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. The tripartite genome of this zoonotic virus is capable of reassortment and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We have developed an in vitro virus-like particle production assay for Oropouche virus, allowing us to study the assembly and release of this dangerous pathogen without high-containment biosecurity. We determined the polyprotein sites that are cleaved to yield the mature Oropouche virus surface glycoproteins and characterised the cellular machinery required for glycoprotein secretion. Our study provides important insights into the molecular biology of Oropouche virus infection, in addition to presenting a robust virus-like particle production assay that should facilitate future functional and pharmacological inhibition studies.
Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by virus-like particle
