Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun 29;97(6):e0043323.
doi: 10.1128/jvi.00433-23. Epub 2023 Jun 6.

Structures of Langya Virus Fusion Protein Ectodomain in Pre- and Postfusion Conformation

Aaron J May  1   2 , Karunakar Reddy Pothula  1 , Katarzyna Janowska  1 , Priyamvada Acharya  1   2   3
Affiliations

Structures of Langya Virus Fusion Protein Ectodomain in Pre- and Postfusion Conformation

Aaron J May et al. J Virol. .

Abstract

Langya virus (LayV) is a paramyxovirus in the Henipavirus genus, closely related to the deadly Nipah (NiV) and Hendra (HeV) viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-electron microscopy (cryo-EM) structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and postfusion conformations. The LayV-F protein exhibits pre- and postfusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and postfusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide (FP) is buried within a highly conserved, hydrophobic interprotomer pocket in the prefusion state and is notably less flexible than the rest of the protein, highlighting its "spring-loaded" state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to postfusion conversion that may apply more broadly to paramyxoviruses. IMPORTANCE The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which have important vaccine and therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to the Paramyxoviridae family.

Keywords: conformational change; cryo-EM; fusion peptide; fusion protein; glycoprotein; henipavirus; paramyxovirus; structural biology; viral entry; viral protein.

PubMed Disclaimer

Conflict of interest statement

The authors declare a conflict of interest. A.J.M. and P.A. have submitted a patent application covering Henipavirus F protein modifications based on this study. May A, Acharya P., U.S. Provisional Patent Application No. 63/458,400 filed 4/10/2023.

Figures

FIG 1
FIG 1
Structures of LayV-F in pre- and postfusion conformation. Cryo-EM maps of the pre- and postfusion conformations of LayV-F along with the fitted models with a sequence key. (A and B) Cryo-EM reconstructions of LayV-F in prefusion (A) and postfusion (B) conformations, colored by protomer. Map regions colored in different shades of blue (prefusion) and yellow (postfusion) for each protomer. Representative reference-free 2D classes used for the reconstruction are displayed above each map. (C and D) Cartoon representation of atomic models of LayV-F in prefusion (C) and postfusion (D) conformations that were built into the maps shown in panels A and B. In each, one protomer is colored by domain, based on the color scheme in panel E, and the other two protomers are colored gray. (E) Sequence domain key for LayV-F. SS, secretion signal, cleaved prior to purification. DI, DII, and DIII, domains I, II, and III, respectively; FP, fusion peptide; HRA and HRB, heptad repeats A and B, respectively; foldon, trimerization domain; TS, Twin-Strep tag, used for purification with modified streptavidin resin.
FIG 2
FIG 2
Role of conserved cysteines during conformational conversion. Demonstration of the role of conserved cysteines in each domain in pre- to postfusion conformational changes. (A) Zoomed-in view of the DIII domain, including the fusion peptide (orange) and HRA (yellow), shown for the prefusion (left) and postfusion (right) structures. Color scheme continued from Fig. 1E. The cleavage site at R104 is identified, as well as the disulfide bond formed by C66 and C187 (yellow sticks). This disulfide bond is a hinge point around which the HRA straightens into the central coiled-coil in the postfusion F structure. (B and C) Superimposition of pre- and postfusion conformation domains DI (B) and DII (C). Color scheme continued from Fig. 1E, with the postfusion conformation colored in a lighter shade. Disulfide bonds are shown as yellow sticks and labeled. For DII, the movement of the HRB linker region is indicated by an arrow. (D) Sequence alignment of paramyxovirus fusion proteins in the regions surrounding conserved cysteines, with cysteine residue numbers and domains labeled. Viruses colored blue are from the Henipavirus genus and colored tan for other paramyxoviruses. Residues are colored by type per MView standard (51). Blue, alcohol; green, hydrophobic; dark blue, negative charge; red, positive charge; purple, polar; yellow, cysteine.
FIG 3
FIG 3
Fusion peptide burial and conservation. (A) Exploration of motion in the pre-F structure through B factor and DDM analysis, with a focus on the features and sequence conservation of the fusion peptide pocket. (A) Structure of the prefusion LayV-F protein shown in the cartoon representation and colored by B factors. (B) Difference distance matrix plot comparing relative residue positions between prefusion and postfusion conformation. Sequence key from Fig. 1E included along each axis. Gray areas in the bottom right half of the plot are the result of sequences not present in the postfusion model. (C) Hydrophobic fusion peptide pocket in the prefusion state, with fusion peptide depicted as orange cartoon and surface electrostatic view of the pocket. (D) Side chain stick view of the fusion peptide pocket, with hydrophobic residues involved shown with side chains as green sticks. (E) Cartoon representations of a single prefusion-state protomer, colored by sequence conservation across Henipavirus fusion proteins. Color scale generated by ChimeraX default AL2CO entropy measure. (F) Zoomed-in view of the fusion peptide pocket, colored by sequence conservation as in E. The fusion peptide pocket is among the most strongly conserved regions of the protein.
FIG 4
FIG 4
Structure and antigenicity comparison of Henipavirus fusion proteins. Comparison of LayV-F structures, surface residue types, and glycosylation patterns of previously determined NiV-F and HeV-F structures, with a focus on important antigenic sites. (A) Superimposition of a single prefusion protomer of LayV-F (blue), NiV-F (cyan), and HeV-F (green) in cartoon representation. PDB accession numbers are 8FEJ for LayV-F, 5EVM for NiV-F, and 5EJB for HeV-F (15, 24). (B) Zoomed-in view of a loop within the folded HRA, one of the few areas of significant structural difference between LayV-F and NiV/HeV-F. (C) Binding site of known paramyxovirus F protein-neutralizing antibodies, labeled by the species targeted. NiV-F trimer from the 12B2-bound structure shown as cartoon and antibody Fabs shown in surface view. Only one Fab per type shown; all except PIA174 bind with 3:1 stoichiometry. PDB accession numbers are 6MJZ for PIA174, 7KI4 for 12B2, 6T3F for mAb66, 7KI6 for 1F5, and 6TYS for 5B3 (25, 30, 52). (D) Surface view of LayV-F and NiV-F, with glycosylation sites (residues meeting the N-X-S/T glycosylation sequence) colored teal. (E) Surface representation of LayV-F colored by sequence conservation across Henipavirus fusion proteins. Color scale generated by ChimeraX default AL2CO entropy measure; magenta indicates high conservation, and teal indicates high variability (53). (F) Surface representation of paramyxovirus fusion proteins, showing only main chain atoms, colored by residue type and shown from top-down view. Tan, hydrophobic; pink, polar; red, negative; blue, positive; teal, potential glycosylation. PIV5, PBD accession no. 4GIP (23).
FIG 5
FIG 5
Fusion-triggering hypothesis for paramyxoviruses. Proposed mechanism of Henipavirus fusion triggering based on new data and previous knowledge. (A) Existing model of Henipavirus (HNV) fusion protein triggering based on Liu et al. (36). HNV-G, brown, shown in a two-receptor binding domain down “closed” state, bound to HNV-F in prefusion conformation. Exposure to ephrin B2 and B3 receptors induces opening of HNV-G, triggering conversion of HNV-F from prefusion to the prehairpin state. The triggering interaction is labeled with a star. (B) Hypothesized structural basis for the G-F interaction shown in panel A, wherein a two-step mechanism involving disruption of the fusion peptide burial pocket through the action of the HNV-G stalk on DII frees the fusion peptide, allowing conformational conversion. The figure was made using resources from BioRender.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zhang X-A, Li H, Jiang F-C, Zhu F, Zhang Y-F, Chen J-J, Tan C-W, Anderson DE, Fan H, Dong L-Y, Li C, Zhang P-H, Li Y, Ding H, Fang L-Q, Wang L-F, Liu W. 2022. A zoonotic henipavirus in febrile patients in China. N Engl J Med 387:470–472. doi:10.1056/NEJMc2202705. - DOI - PubMed
    1. Wu Z, Yang L, Yang F, Ren X, Jiang J, Dong J, Sun L, Zhu Y, Zhou H, Jin Q. 2014. Novel Henipa-like virus, Mojiang paramyxovirus, in rats, China, 2012. Emerg Infect Dis 20:1064–1066. doi:10.3201/eid2006.131022. - DOI - PMC - PubMed
    1. Lee S-H, Kim K, Kim J, No JS, Park K, Budhathoki S, Lee SH, Lee J, Cho SH, Cho S, Lee G-Y, Hwang J, Kim H-C, Klein TA, Uhm C-S, Kim W-K, Song J-W. 2021. Discovery and genetic characterization of novel paramyxoviruses related to the genus henipavirus in Crocidura species in the Republic of Korea. Viruses 13:2020. doi:10.3390/v13102020. - DOI - PMC - PubMed
    1. Field H, Young P, Yob JM, Mills J, Hall L, Mackenzie J. 2001. The natural history of Hendra and Nipah viruses. Microbes Infect 3:307–314. doi:10.1016/s1286-4579(01)01384-3. - DOI - PubMed
    1. Rima B, Balkema-Buschmann A, Dundon WG, Duprex P, Easton A, Fouchier R, Kurath G, Lamb R, Lee B, Rota P, Wang L, Ictv Report Consortium . 2019. ICTV virus taxonomy profile: Paramyxoviridae. J Gen Virol 100:1593–1594. doi:10.1099/jgv.0.001328. - DOI - PMC - PubMed

Publication types

Supplementary concepts

LinkOut - more resources

-