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Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage.

ABSTRACT: The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are lethal emerging paramyxoviruses. EphrinB2 and ephrinB3 have been identified as receptors for henipavirus entry. NiV and HeV share similar cellular tropisms and likely use an identical receptor set, although a quantitative comparison of receptor usage by NiV and HeV has not been reported. Here we show that (i) soluble NiV attachment protein G (sNiV-G) bound to cell surface-expressed ephrinB3 with a 30-fold higher affinity than that of sHeV-G, (ii) NiV envelope pseudotyped reporter virus (NiVpp) entered ephrinB3-expressing cells much more efficiently than did HeV pseudotyped particles (HeVpp), and (iii) NiVpp but not HeVpp entry was inhibited efficiently by soluble ephrinB3. These data underscore the finding that NiV uses ephrinB3 more efficiently than does HeV. Henipavirus G chimeric protein analysis implicated residue 507 in the G ectodomain in efficient ephrinB3 usage. Curiously, alternative versions of published HeV-G sequences show variations at residue 507 that can clearly affect ephrinB3 but not ephrinB2 usage. We further defined surrounding mutations (W504A and E505A) that diminished ephrinB3-dependent binding and viral entry without compromising ephrinB2 receptor usage and another mutation (E533Q) that abrogated both ephrinB2 and -B3 usage. Our results suggest that ephrinB2 and -B3 binding determinants on henipavirus G are distinct and dissociable. Global expression analysis showed that ephrinB3, but not ephrinB2, is expressed in the brain stem. Thus, ephrinB3-mediated viral entry and pathology may underlie the severe brain stem neuronal dysfunction seen in fatal Nipah viral encephalitis. Characterizing the determinants of ephrinB2 versus -B3 usage will further our understanding of henipavirus pathogenesis.

SUBMITTER: Negrete OA

PROVIDER: S-EPMC2045465 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage.

Negrete Oscar A OA Chu David D Aguilar Hector C HC Lee Benhur B

Journal of virology 20070725 19

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are lethal emerging paramyxoviruses. EphrinB2 and ephrinB3 have been identified as receptors for henipavirus entry. NiV and HeV share similar cellular tropisms and likely use an identical receptor set, although a quantitative comparison of receptor usage by NiV and HeV has not been reported. Here we show that (i) soluble NiV attachment protein G (sNiV-G) bound to cell surface-expressed ephrinB3 with a 30-fold higher affinity than that ...[more]

PMID: 17652392

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Project description:Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

Project description:Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic paramyxovirus which belongs to Henipavirus family, causes severe respiratory disease, and may lead to fatal encephalitis infections in humans. NiV and HeV glycoproteins (G) bind to the highly conserved human ephrin-B2 and B3 (EFNB2 & EFNB3) cell surface proteins to mediate the viral entry. In this study, various molecular modelling approaches were employed to understand protein-protein interaction (PPI) of NiV and HeV glycoprotein (84% sequence similarity) with Human EFN (B2 and B3) to investigate the molecular mechanism of interaction at atomic level. Our computational study emphasized the PPI profile of both the viral glycoproteins with EFN (B2 and B3) in terms of non-bonded contacts, H-bonds, salt bridges, and identification of interface hotspot residues which play a critical role in the formation of complexes that mediate viral fusion and entry into the host cell. According to the reports, EFNB2 is considered to be more actively involved in the attachment with the NiV and HeV glycoprotein; interestingly the current computational study has displayed more conformational stability in HeV/NiV glycoprotein with EFNB2 complex with relatively high binding energy as compared to EFNB3. During the MD simulation, the number of H-bond formations was observed to be less in the case of EFNB3 complexes, which may be the possible reason for less conformational stability in the EFNB3 complexes. The current detailed interaction study on the PPI may put a path forward in designing peptide inhibitors to obstruct the interaction of viral glycoproteins with host proteins, thereby inhibiting viral entry.Graphical abstractThe viral attachment and fusion of Nipah and Hendra virus was explored through the interaction between viral glycoprotein and the host cell surface ephrin protein. The MD simulation results displayed more stability in Nipah and Hendra glycoprotein with EFNB2 as compared to EFNB3. The residue Glu533 in the central cavity of HeV/NiV glycoprotein protein identified as the potential hotspot in binding with the G-H loop of EFNB2.Supplementary informationThe online version contains supplementary material available at 10.1007/s12039-022-02110-9.

Project description:UnlabelledHendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.ImportanceViral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.

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Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage.

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Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage.

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