The orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway that is specific for the insect host
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The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises several important pathogens including the human-infecting Oropouche virus and animal-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein spikes, with Gc head and stalk domains forming distinctive tripods covering the envelope-proximal Gn. Spike ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments to mediate virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from animals bearing head/stalk deletions. However, orthobunyavirus isolations from arthropods in nature appear to maintain these domains strictly.
To investigate this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-Δ7). In mammalian cells BUNV-WT and BUNV-Δ7 grew to equivalent titres, whereas BUNV-Δ7 titres from insect cells were 1000-fold lower than BUNV-WT and strikingly produced no virions following blood meal infection of Aedes mosquitoes.
To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-Δ7 Gc and NP components were equivalent, suggesting Δ7-Gc was assembly-deficient. To explore this, we investigated Gc and Δ7-Gc interactions during BUNV-WT and BUNV-Δ7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing Δ7-Gc exhibited markedly reduced NP interactions in insect cells. We propose Gc recruits genome segments during virion assembly and that the complete Gc head/stalk assembly is maintained in nature due to its essential role in the insect host.
IMPORTANCE
Orthobunyaviruses are arthropod-borne viruses that cause severe disease in humans and animals, including congenital malformations and abortions. Orthobunyavirus Gn and Gc glycoproteins form spikes, which mediate entry and genome recruitment during assembly. In nature, OBVs bearing large deletions within Gc head/stalk domains have been isolated from animals, yet the head/stalk domains appear to be strictly maintained within insects. To investigate this discrepancy, we compared the multiplication of Bunyamwera orthobunyavirus (BUNV-WT) with head/stalk-deleted variant (BUNV-Δ7). In mammalian cells both viruses reached similar titres, but strikingly BUNV-Δ7 failed to produce virions in both insect cells and Aedes mosquitoes. We showed this was because BUNV-Δ7 failed to assemble new virions, revealing its head/stalk-deleted Gc was deficient in interactions with genome components. We propose that Gc drives species-specific interactions with genome segments during virion assembly, explaining why Gc head/stalk domains are conserved in nature due to their essential role in the insect host.
