Headless Henipaviral Receptor Binding Glycoproteins Reveal Fusion Modulation by the Head/Stalk Interface and Post-receptor Binding Contributions of the Head Domain

Abstract

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Henipavirus zoonosis: outbreaks, animal hosts and potential new emergence

“…Different characteristics among closely related viruses can be utilized in comparative functional investigations to understand aspects of henipavirus biology such as evolution, host tropism, cross-species transmission and pathogenicity. So far CedV ( Marsh et al, 2012 ; Lieu et al, 2015 ; Laing et al, 2018 , 2019 ; Schountz et al, 2019 ; Yeo et al, 2021 ), KV ( Pernet et al, 2014a ; Lee et al, 2015 ) and MojV ( Rissanen et al, 2017 ; Cheliout Da Silva et al, 2021 ) have been involved in a number of such studies. Non-pathogenic henipa-like viruses such as CedV could also be used as immunogen vectors in vaccines against pathogenic henipavirus disease, since antigenic overlap could induce cross-reactivity between closely related viruses.…”

Structures of Langya virus fusion protein ectodomain in pre and post fusion conformation

“…Within the Henipavirus and Morbilivirus genera, the attachment and fusion proteins are believed to form complexes prior to receptor engagement (33) (Figure 5A). While this might suggest a mechanism where the attachment protein functions as a clamp on the fusion protein, maintaining the pre-fusion state before receptor binding, studies assaying fusion competency have demonstrated that the fusion protein alone is not sufficient for virus and host cell fusion, indicating that fusion triggering involves a more complex mechanism of interaction between the two proteins (9,48).…”

Drivers and Distribution of Henipavirus-Induced Syncytia: What Do We Know?

“…Interactions with the cytoskeleton also modulate cell-cell fusion, as highlighted by proteomics [61]. The study of HNV mutants also revealed that the early and late steps of the membrane fusion cascade are modulated by several regions of G head and stalk domains [31,32,48] and of F [62,63]. Furthermore, N-and O-glycans in most paramyxoviral glycoproteins have been shown to modulate protein expression, folding, and transport; for HNVs in particular, several N-and O-glycans in NiV and HeV G and F modulate virus-cell and cell-cell fusion independently of protein expression [64][65][66].…”

“…Although a wide range of cell lines are susceptible to HNV-induced cell-cell fusion and qualitative patterns (susceptible versus refractory to cell-cell fusion) seem conserved across HNVs, quantitative variations have been reported. For instance, CedV induces significantly less cell-cell fusion than NiV in human kidney HEK293T cells [48]. Similarly, KuV and MojV also appear to form fewer syncytia than NiV in HEK293T and hamster kidney BHK cells [23,57].…”

“…Studies of the cascade of interactions between G, F, and ephrin receptors are dominated by in vitro experiments using cells transfected with wild-type or mutant G and F and measuring cell-cell fusion as an output (Box 1; Figure 2) [22,[31][32][33]47]. Briefly, the binding of G to a compatible ephrin first induces conformational changes in G that expose a sequestered F-triggering region on its stalk domain, which then triggers the unfolding of F [31,32,48]. The fusion peptide of F anchors onto the target cell membrane and adopts a pre-hairpin intermediate conformation.…”