Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope

Significance

Bunyaviruses are emerging zoonotic pathogens of public-health concern. Lack of structures for proteins on the viral membrane (“envelope”) surface limits understanding of entry. We describe atomic-level structures for the globular “head” of the envelope protein, glycoprotein N (Gn), from two members, severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV), of Phleboviruses genus in the bunyavirus family, and a structure of the SFTSV Gn bound with a neutralizing antibody Fab. The results show the folded Gn structure and define virus-specific neutralizing-antibody binding sites. Biochemical assays suggest that dimerization, mediated by conserved cysteines in the region (“stem”) connecting the Gn head with the transmembrane domain, is a general feature of bunyavirus envelope proteins and that the dimer is probably the olimeric form on the viral surface.

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae. Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4–5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

The Bunyaviridae is a large family of human, animal, and plant pathogens spanning five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus (1). With the exception of hantaviruses, all other bunyaviruses are arthropod-borne viruses. Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) belong to the Phlebovirus genus, which can cause emerging infectious diseases in humans (1–3), as emphasized by the recent imported case of RVFV infection in China after its first emergence in Africa over 80 y ago (4).

SFTS cases were first reported in China during 2007; however, the causative agent was not isolated from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction until 2011 (2, 5). SFTSV-infected patients have been found in at least 13 provinces in China, with a case fatality rate of ≈12%. In 2013, cases of SFTS were reported in South Korea and Japan, with a case fatality rate of 35.4% and 50%, respectively (6–8). Unfortunately, vaccine or antiviral intervention against SFTSV remain unavailable.

RVFV is an emerging mosquito-borne zoonotic infectious pathogen and the prototype virus of the Phlebovirus genus (1). RVFV was isolated in Kenya in 1930 (9, 10). Recurring outbreaks of RVFV disease have been reported in ruminants and humans in Africa and the Arabian Peninsula (11, 12), constituting a significant threat to global public health and agriculture. Humans can be infected by bites from virus-carrying mosquitoes or through contact with bodily fluids of the infected animals (13). RVF patients display a self-limiting febrile illness, and some cases may develop lethal hemorrhagic fever, neurologic disorders, or blindness (13). Vaccines have been used to control RVF among livestock in endemic regions in Africa and the Arabian Peninsula (14, 15). However, formalin-inactivated whole-virus vaccines show little immunogenicity (16, 17), whereas live-attenuated vaccine is teratogenic in pregnant sheep and cattle (18, 19). Currently, there are no effective vaccines or antiviral agents approved for use in humans.

As with the other members of the Bunyaviridae family, SFTSV and RVFV genomes contain three negative-stranded RNA segments (L, M, and S) (1). The M segment encodes a glycoprotein precursor, which can be cleaved by cellular proteases during translation (20). For SFTSV, the precursor can be processed into two subunits: glycoprotein N (Gn) and glycoprotein C (Gc) (1), while for RVFV, one more subunit, called nonstructural protein in the M segment is processed, in addition to Gn and Gc. Gn/Gc are responsible for attachment and membrane fusion, which is required for host cell entry (21–23). The recently solved crystal structures of RVFV and SFTSV Gc proteins revealed architectural similarity with class II viral fusion proteins (24, 25). Low pH can induce RVFV Gc oligomerization, but has no influence on Gn (23). Recent studies reveal that the lectin dendritic-cell (DC) specific intercellular adhesion molecule 3-grabbing nonintegrin (SIGN) is identified as the entry factor required for many phleboviruses, including SFTSV, RVFV, Toscana virus (TOSV), and Uukuniemi virus (UU.K.V) (26, 27). L-SIGN, another C-type lectin, shares 77% sequence homology with DC-SIGN and acts as an attachment receptor for these phleboviruses, rather than as an endocytic receptor (28). Nonmuscle myosin heavy chain II A (NMMHC-IIA) is reported as a critical factor contributing to the efficiency of early SFTSV infection, and the recombinant Gn protein is capable of binding NMMHC-IIA, indicating that Gn is likely the key receptor-binding protein (26).

Gn and Gc are two major antigenic components on the viral surface and are the targets of specific neutralizing antibodies (29). MAb 4–5 is a human-origin, neutralizing monoclonal antibody targeting Gn, showing cross-neutralizing activity to a wide range of SFTSV isolates in China (30). An RVFV Gn/Gc subunit vaccine was previously shown to elicit a strong neutralizing antibody response in sheep (31).

Here, we report the crystal structures of two Gn head domains (from SFTSV and RVFV) and the SFTSV Gn head domain in complex with MAb 4–5, a neutralizing antibody identified in SFTS recovered patients (30). These two Gn head domains display a similar overall configuration but a different overall topology to the Gn head structure of Puumala hantavirus (PUUV) (32). Four cysteine residues highly conserved in the stem region of Gn are responsible for dimerization, suggesting the Gn cysteine-mediated dimer model might apply to the entire Bunyaviridae family. The complex structure reveals that the key residues in SFTSV recognized by neutralizing MAb 4–5 is not conserved in RVFV; therefore, MAb 4–5 cannot bind to RVFV Gn. Moreover, conserved exposure amino acid alignment of 11 members in Phlebovirus genus indicates that domain III of Gn provide epitopes for specific neutralizing antibody, while domain II is probably an ideal region recognized by a broadly neutralizing antibody. Altogether our findings have proved that Gn is a promising antigen for vaccine development and the crystal structures provide a molecular basis for the rational design of vaccines and antiviral drugs.

Results

Overall Structure of the Gn Head Domain: SFTSV and RVFV.

Both SFTSV and RVFV Gn are type I transmembrane proteins with the N-terminal ectodomain binding on the cell surface and a C-terminal transmembrane helix anchored on the virus membrane (Fig. 1A). The ectodomain can be divided into the head and stem domains. To facilitate crystallization, the stem region of SFTSV Gn was removed by limited trypsin digestion due to an unsuccessful effort with the full-length ectodomain for crystallization. The last amino acid visible in the crystal structure is N340. However, the molecular weight measured by mass spectrometry (MS) indicates that the cleavage site is between residues K371 and S372, suggesting a conformational disorder in the C terminus of the truncated Gn (residues 340–372), instead of degradation during crystallization (Fig. 1A). We designed the construct of the RVFV Gn head domain based on the structure of the SFTSV Gn head domain, and succeeded in obtaining its crystal structure. Both the crystal structures of SFTSV and RVFV Gn head domains were determined at a resolution of 2.6 Å (Table S1). A Dali search within the Protein Data Bank (PDB) failed to identify any existing structures to the Gn of both SFTSV and RVFV, suggesting a novel fold. The structure of SFTSV Gn was solved by “antibody-walking” (Materials and Methods), in which the antibody provides model-based phasing information to determine the unknown structure. The RVFV Gn structure was solved by molecular replacement, using SFTSV Gn as a search model.

Fig. 1.

Overview of the Gn structures in SFTSV and RVFV. (A) Schematic representation of the full-length SFTSV and RVFV Gn proteins. For SFTSV, subdomain I is in hot pink, subdomain II is in marine, and subdomain III is in green. The unstructured part is in diagonal strips. Signal peptide (SP), transmembrane anchor (TM), and cytoplasmic tail (CT) are in light gray, medium gray, and dark gray, respectively. Free cysteines and disulfide bonds are labeled accordingly and the stem region is indicated. Glycans are linked to N33 and N63, respectively. For RVFV, subdomain I is in light pink, subdomain II is in pale cyan, and subdomain III is in limon, respectively. A predicted N-linked glycan not observed in the structure is denoted with an open square. All other regions (SP, TM, CT, and stem region) are depicted as in SFTSV. (B) Cartoon representation of the SFTSV Gn head structure, with disulfide bonds in orange stick and glycans in gray sphere. (C) Cartoon representation of the RVFV Gn head structure, with disulfide bonds in orange stick. (D) Topology diagram of the SFTSV Gn head domain following the same coloring scheme as in the cartoon representation, with detailed secondary structure elements and disulfide bonds (dashed lines labeled with SS). The glycosylation sites are labeled in gray balls. (E) Topology diagram of the RVFV Gn head domain following the same coloring scheme as in the cartoon representation, with detailed secondary structure elements and disulfide bonds (dashed lines labeled with SS).

Table S1.

Crystallographic data collection and refinement statistics

Parameter*	SFTSV Gn head	RVFV Gn head SAD	RVFV Gn head Native	SFTSV Gn head-MAb 4–5
Data collection statistics
Wavelength	0.97853	1.03906	0.97853	0.97905
Resolution, Å	50–2.6 (2.69–2.60)	50–3.4 (3.52–3.40)	50–2.5 (2.59–2.50)	50–2.1 (2.18–2.10)
Space group	I4	P6522	P212121	P212121
Cell dimensions
a, b, c, Å	87.41, 87.41, 91.00	97.18, 97.18, 184.13	51.571, 73.874, 98.125	82.61, 103.19, 112.03
α, β, γ, °	90, 90, 90	90, 90, 120	90, 90, 90	90, 90, 90
Unique reflections	10,777 (1,108)	7,634 (740)	13,526 (1,286)	56,517 (5,372)
Rmerge†, %	7.9 (84.8)	11.7 (88.7)	10.4 (77.4)	8.8 (28.1)
I/σI	15.5 (1.7)	23.5 (3.8)	16.8 (1.8)	22.7 (7.1)
Completeness, %	96.3 (100)	99.9 (100)	99.5 (96.5)	99.5 (95.8)
Redundancy	4.2 (4.6)	14.8 (15.4)	6.7 (5.8)	8.0 (4.7)
Wilson B-factor	79.77	62.5	34.5	23.04
Refinement
Resolution, Å	36.6–2.60	50–3.40	50–2.50	38.7–2.10
Rwork/Rfree, %	23.41/29.08	26.10/30.85	19.67/24.83	17.51/20.71
No. atoms
Protein	2,450	2,223	2,437	5,776
Ligand/ion	39	6	0	67
B-factors
Protein	100.15	46.28	38.59	27.35
Rmsd
Bond lengths, Å	0.003	0.013	0.015	0.005
Bond angles, °	0.777	1.54	1.78	1.040
Ramachandran plot, %
Favored region	95	95	94	96.78
Allowed region	4.05	3.6	6.4	2.95
Outliers region	0.95	1.4	0.0	0.27

^*Values in parentheses are for the highest-resolution shell.

^†R_merge = Σhkl |I-<I>|/ΣhklI, where I is the intensity of unique relfection hkl and <I> is the average over symmetry-related observations of unique reflection hkl.

The Gn head domains of both SFTSV and RVFV fold into a similar triangular shape architecture consisting of three subdomains (Fig. 1 B and C). Subdomains I and II form the foundation bed supporting the subdomain III protruding on the top (Fig. 1 B and C and Fig. S1A). However, the three subdomains show a different arrangement between these two viruses, with an average rmsd of 5.010, 2.393, and 1.825 for subdomains I, II, and III, respectively (Fig. S1B). Specifically, in subdomain I of SFTSV, a five-stranded β-sheet (β2, β3, β4, β5, and β9), one helix (α3), and one 3₁₀-helix (η2) are located on the interface of subdomains I and II. Two α-helices (α1 and α2), three 3₁₀-helices (η1, η3, and η4), and two sets of small antiparallel β-strands (β1 and β8, β6 and β7) flank the β-sheet on the opposite side. A free cysteine (C99) on α3 is located in the interior of subdomain I. For the RVFV Gn subdomain I, a four-stranded β-sheet (β1, β2, β3, and β6) and three α-helices (α2, α3, and α4) can be found on the interface of subdomains I and II. One α-helix (α1), three 3₁₀-helices (η1, η2, and η3), and one pair of small antiparallel β-strand (β4 and β5) are on the opposite side. β-Strands are the major component in subdomain II and the pattern of β-strands between SFTSV and RVFV is the same (Fig. 1 D and E and Fig. S2). The core structure comprising the six β-strands is located next to subdomain I and a three-stranded β-sheet connects to subdomain III. The only different secondary element in subdomain II between these two is the α-helix. SFTSV contains an α4, whereas RVFV does not have it in subdomain II. The secondary elements in subdomain III between SFTSV and RVFV are similar. Four β-strands and three α-helices stabilize subdomain III, in which α5 in SFTSV is replaced by η5 in RVFV (Fig. 1 D and E). Although the Gn structures in the Phlebovirus genus display similar configurations, the overall structures between the Phlebovirus genus and Hantavirus genus are distinct (Fig. S1 C and D). Furthermore, the topology between these two genera is completely different (Fig. S1E).

Fig. S1.

Comparison of Gn head structures among RVFV, SFTSV, and PUUV. (A) Superimposition of the Gn head domain between RVFV and SFTSV. The structures are shown in cartoon with the colors in agreement with Fig. 1. (B) Three subdomains comparison between RVFV and SFTSV. (C) Comparison among SFTSV Gn, RVFV Gn, and PUUV Gn head domains. (D) Topology diagram of PUUV Gn, with the glycosylation sites labeled in gray balls.

Fig. S2.

Secondary structure and amino acid alignment of the Gn ectodomain between SFTSV and RVFV. The sequences are aligned based on a pairwise comparison of the corresponding crystal structures. Conserved residues are highlighted in red. Subdomain colors in head are labeled consistent with Fig. 1A. The invisible part of the Gn head structure is indicated in light gray. The stem region is defined in dark gray. Paired cysteines are labeled below sequence (RVFV in blue and SFTSV in green). Two cysteines that are important to Gn stability are labeled with blue asterisks. Four cysteines that are responsible for Gn dimerization are labeled with purple asterisks.

The glycosylation sites are observed to be different between SFTSV and RVFV Gn. For SFTSV, two N-linked glycans (N33 and N63) can be observed in subdomain I (Fig. 1 A, B, and D and Fig. S2), which is consistent with theoretical predictions. For RVFV, although N438 is predicted to be an N-linked glycosylation site, no glycans can be observed in the solved crystal structure in the insect cell-expressed protein (Fig. 1 A, C, and E).

Both the SFTSV and RVFV Gn are cysteine-rich proteins. SFTSV Gn has 27 cysteines, whereas RVFV Gn has 28 cysteines. Twelve cysteines in the head domains are identical between these two Gn, and all of the other 10 cysteines in the stem domains are conserved for the 11 sequences of the available phleboviruses (Figs. S2 and S3A). The SFTSV Gn head domain contains eight disulfide bonds and a free cysteine. Specifically, two disulfide bonds (C26-C49 and C143-C156) and an unpaired cysteine (C99) are in subdomain I, one disulfide bond is in subdomain II (C206-C216), one disulfide bond is across subdomains II and III (C180-C327), and four disulfide bonds are in subdomain III (C258-C305, C266-303, C274-C280, and C287-C292). The RVFV Gn head domain is stabilized by nine disulfide bonds, four of which are in subdomain I (C179-C188, C229-C239, C250-C281, and C271-C284), one of which is in subdomain II (C322-C332), three of which are in subdomain III (C374-C434, C402-C413, and C420-C425), and one of which is across subdomains II and III (C304-C456). Six disulfide bonds (C271-C284 in subdomain I, C304-C456 in subdomain II, C322-C332 crossing subdomain II and III, C374-C434, C402-C413, and C420-C425 in subdomain III, RVFV Gn numbering) are conserved between the SFTSV and RVFV Gn head domains (Fig. S2).

Fig. S3.

Amino acid sequence alignment of the Gn stem region in Phlebovirus, Nairovirus, and Hantavirus genera of the Bunyavirus family. Residue numberings are based on the full-length proteins. Conserved cysteines are labeled with blue asterisks. (A) Amino acid sequence alignment of the Gn stem region in Phlebovirus genus. Database sequence accession numbers: RVFV, gb: YP_003848705.1; SFTSV, gb: AFB82725.1; HRTV, gb: AIF75092.1; Candiru virus, gb: YP_004347992.1; PTV, gb: AAA47110.1; Chagres virus, gb: AEL29641.1; Sandfly fever Naples virus (SFNV), gb: AIS25027.1; TOSV, gb: ABZ85665.1; Uukuniemi virus (UUKV), gb: NP_941979; Bhanja virus, gb: YP_009141014.1; and Palma virus, gb: AGC60100.1. Two cysteines that are important to Gn stability are labeled with yellow asterisks. Four cysteines that are responsible for Gn dimerization are labeled with purple asterisks. (B) Amino acid sequence alignment of the Gn stem region in Nairovirus genus. Database sequence accession nos.: Crimean-Congo hemorrhagic fever nairovirus (CCHFV), gb: AAM48107.1; Bandia virus, gb: AMT75384.1; Dera Ghazi Khan nairovirus (DGK virus), gb: AMT75390.1; Dugbe nairovirus, gb: AAA42974.1; Hughes nairovirus, gb: AMT75408.1; Sakhalin nairovirus, gb: AMT75420.1; Thiafora nairovirus, gb: ALD84356.1. (C) Amino acid sequence alignment of the Gn stem region in Hantavirus genus. Database sequence accession nos.: PUUV, gb: CAB43026.1; Tula virus (TULV), gb: NP_942586.1; Adler hantavirus, gb: AIY68299.1; Hantaan hantavirus (HTNV), gb: AFS64897.1; Andes hantavirus (ANDV), gb: AAO86638.1; Bayou hantavirus (BAYV), gb: ADE10201.1; Blue River virus, gb: AAC03793.1; Catacamas virus: gb: ABA39272.1; Dobrava-Belgrade hantavirus (DOBV), gb: AAN75012.1; Fugong virus (FUGV), gb: AMB43173.1; Gou virus, gb: AGC97080.1; Jeju virus, gb: AEX56232.1; Kenkeme virus (KKMV), gb: AIL25322.1; Khabarovsk hantavirus (KHAV), gb: AIL25316.1; Muju virus, gb: AGE47781.1; Oran virus, gb: AAB87910.1; Prospect Hill hantavirus (PHV), CAA38922.1; Rio Mamore hantavirus (RIOMV), gb: ACU46022.1; Sangassou hantavirus (SANGV), gb: AEZ02947.1; Seoul hantavirus, gb: AFS64904.1; Sin Nombre hantavirus (SNV), gb: AIA08876.1; Topografov hantavirus (TOPV), gb: CAB42098.1.

Dimerization of Gn Through Its Stem Region.

The full-length SFTSV Gn ectodomain was produced using the baculovirus expression system. Results from size-exclusion chromatography using a Superdex 200 10/300 GL column showed the peaks eluted at 12.6 mL and 14.4 mL, corresponding to the dimer and monomer in solution, respectively (Fig. 2A). Nonreducing SDS/PAGE shows that the dimer is linked by disulfide bonds (Fig. S4A). Notably, the dimer was completely disassociated after a treatment of limited trypsin proteolysis (Fig. 2A and Fig. S4A), indicating that the C terminus of Gn is solely responsible for dimerization via disulfide bonds (Fig. 2A). To characterize the C terminus of Gn, we constructed the unstructured part of Gn (Gn-C), which corresponds to residues 338–452. The Gn-C protein forms a dimer under nonreducing conditions, which is consistent with the above-described result (Fig. S4B). Full-length RVFV Gn forms a dimer under nonreducing conditions as well (Fig. S4C).

Fig. 2.

The C terminus of SFTSV Gn is responsible for dimerization. (A) Size-exclusion analysis of the SFTSV Gn monomer and dimer before and after trypsin digestion, indicating that the full-length of the Gn ectodomain has two states, monomer and dimer (black), while the Gn head is monomer only (blue and red). (B) Size-exclusion analysis of SFTSV Gn proteins of wild-type (black), Gn-C430AC447A (red), Gn-C435AC438A (blue), and Gn-C430AC435AC438AC447A (green). 1, SFTSV Gn WT dimer; 2, WT monomer; 3, monomer after typsin digestion; 4, dimer after typsin digestion.

Fig. S4.

SDS/PAGE profile of full-length Gn protein and Gn-C protein of SFTSV. (A) A comparison of the full-length SFTSV Gn protein before and after trypsin digestion under reducing and nonreducing conditions by SDS/PAGE gel with protein standards. The sample numbering is accordance with Fig. 2A. It shows that the full-length Gn dimer is disulfide-bond linked, which display 49 kDa and 98 kDa under a nonreducing condition, respectively. After trypsin digestion, only one band (37 kDa) can be observed under nonreducing condition. (B) SDS/PAGE profile of a SFTSV Gn-C protein under reducing and nonreducing conditions, showing that Gn-C forms dimer under nonreducing conditions, with the molecular weight ≈30 kDa. Gn-C monomer is ≈15 kDa. (C) Western blot analysis of full-length RVFV Gn protein under reducing and nonreducing conditions, indicating that RVFV Gn has dimer form under nonreducing condition, with the molecular mass around 110 kDa.

To determine which cysteines are responsible for dimerization, we used MS and site-directed mutagenesis to analyze Gn-C and the full-length Gn protein of SFTSV. MS analysis identified four intermolecular disulfide bonds involving C430, C435, C438, and C447 from the Gn-C dimer but not the monomer (Table S2), suggesting that these cysteine residues may mediate Gn dimerization. Indeed, simultaneously mutating these four cysteine residues to alanine completely abolished dimer formation of the full-length Gn, leaving only the monomer (Fig. 2B). Furthermore, the C430A/C447A substitution shifted the equilibrium toward the Gn monomer, but failed to abolish the Gn dimer. The C435A/C438A substitution reduced the relative abundance of the dimer more substantially than the C430A/C447A substitution (Fig. 2B), suggesting that although C430, C435, C438, and C447 all contribute to Gn dimerization, the two intermolecular disulfide bonds mediated by C435 and C438 play a major role. MS analysis also identified a disulfide bond between C356 and C424 in both the Gn-C monomer and dimer (Table S2). Mutation of either C356 or C424 severely reduced the yield of the protein (Fig. S5), suggesting that the C356-C424 disulfide bond is critical for stabilizing Gn. The data described above support that the cysteines in the stem region are responsible for dimerization and the cysteines are highly conserved in the same genus (Figs. S3 and S6). It is noteworthy that the last four cysteines (C430, C435, C438, and C447, SFTSV numbering) responsible for dimerization are highly conserved across five genera (Phlebovirus, Hantavirus, Nairovirus, Orthobunyavirus, and Tospovirus) of the Bunyaviridae family, indicating that members in the whole Bunyaviridae likely share the same assembly organization with a Gn disulfide bond-linked dimer (Fig. S7).

Table S2.

Disulfide bonds identified from the Gn-C monomer and dimer

Identified SS bonds	Best E-value (no. peptides or peptide pairs, no. spectra)		Peptide*
	Dimer	Monomer
GN (376, 381)-GN (435, 438)	N.D.	8.77e-11 (5, 54)	SATVCASHFCSSATSGK (5, 10)-DAVDCTFCREFLK (5, 8)
GN (349, 352, 356)-GN (447)	1.29e-04 (1, 2)	2.03e-07 (7, 106)	EVNQPVQRIGQCTGCHLECINGGVRLITLTSELK (12, 15, 19)-NPQCYPAK (4)
GN (424)-GN (447)	6.93e-07 (1, 7)	1.09e-08 (4, 21)	DGTEFTFEGSCMFP (11)-NPQCYPAK (4)
GN (356)-GN (447)	1.90e-07 (1, 8)	1.38e-10 (2, 26)	ECINGGVRLITLTSELK (2)-NPQCYPAK (4)
GN (430)-GN (447)	1.00e-04 (1, 6)	6.81e-07 (2, 24)	NPQCYPAK (4)-DGCDAV (3)
GN (435, 438)	8.86e-26 (1, 19)	5.23e-26 (1, 55)	DCTFCREFLK (2, 5)
GN (349, 352, 356)-GN (424)	2.27e-13 (6, 36)	7.83e-15 (8, 215)	EVNQPVQRIGQCTGCHLECINGGVRLITLTSELK (12, 15, 19)-DGTEFTFEGSCMFP (11)
GN (430, 435, 438, 447)	3.53e-09 (1, 4)	1.47e-12 (1, 46)	DGCDAVDCTFCREFLKNPQCYPAK (3, 8, 11, 20)
GN (430, 435, 438)-GN (447)	1.65e-05 (1, 2)	1.53e-12 (4, 154)	DGCDAVDCTFCREFLK (3, 8, 11)-NPQCYPAKK (4)
GN (376, 381)-GN (376, 381)	1.06e-10 (3, 180)	4.77e-10 (3, 17)	SATVCASHFCSSATSGKK (5, 10)-SATVCASHFCSSATSGK (5, 10)
GN (435, 438, 447)-GN (435, 438, 447)	1.75e-08 (3, 45)	N.D.	DAVDCTFCREFLKNPQCYPAK (5, 8, 17)-DAVDCTFCREFLKNPQCYPAK (5, 8, 17)
GN (356)-GN (424)	5.82e-13 (4, 36)	1.85e-12 (4, 98)	ECINGGVRLITLTSELK (2)-DGTEFTFEGSCMFP (11)
GN (349, 352)	9.33e-44 (1, 63)	9.05e-41 (1, 83)	DGIQEVNQPVQRIGQCTGCHL (16, 19)
GN (376, 381)	6.14e-29 (1, 305)	1.69e-24 (1, 254)	SATVCASHFCSSATSGK (5, 10)
GN (430, 435, 438, 447)-GN (430, 435, 438, 447)	2.85e-07 (2, 25)	N.D.	DGCDAVDCTFCREFLKNPQCYPAK (3, 8, 11, 20)-DGCDAVDCTFCREFLKNPQCYPAK (3, 8, 11, 20)

N.D., not detected.

^*Values are the sites of cysteines in each peptide that are linked by disulfide bonds.

Fig. S5.

C356 and C424 are important to Gn stability. Size-exclusion analysis of wild-type Gn ectodomain (black), Gn ectodomain containing C356A (red), or C424A (blue) on a Superdex 200 gel filtration column.

Fig. S6.

Amino acid sequence alignment of the Gn stem region in Orthobunyavirus and Tospovirus genera of Bunyavirus family. (A) Amino acid sequence alignment of the Gn stem region in Orthobunyavirus genus. Database sequence accession nos.: Bunyamwera virus, gb: AAA42777.1; La Crosse virus, gb: AAA42776.1; Akabane virus, gb: BAC76386.1; Alajuela virus, gb: AIS74643.1; Bwamba virus, gb: AIN37028.1; Capim virus, gb: ALP92389.1; Catu virus, gb: AKO90173.1; Gamboa virus, gb: AIS74639.1; Kairi virus, gb: ABV68909.1; Koongol virus, gb: AKO90176.1; Madrid virus, gb: AGW82139.1; Main Drain virus (MDV), gb: ABV68910.1; Manzanilla virus, gb: AHY22343.1; Marituba virus, gb: AGW82127.1; Nyando virus, gb: AIN37030.1; Oriboca virus, gb: AGW82131.1; Sathuperi virus, gb: BAM15760.1; Shamonda virus, gb: BAM15764.1; Shuni virus, gb: CCH15004.1; Simbu virus, gb: CCG93496.1; Tete virus, gb: AJT55736.1; Wyeomyia virus, gb: AEZ35274.1. (B) Amino acid sequence alignment of the Gn stem region in Tospovirus genus. Database sequence accession nos.: Tomato spotted wilt virus (TSWV), gb: NP_619703.1; Groundnut bud necrosis virus (GBNV), gb: NP_619703; Groundnut ringspot virus (GRSV), gb: AAU10600; Impatiens necrotic spot virus (INSV), gb: AAA46242.1; Watermelon silver mottle virus (WSMV), gb: AAB41723.1; Zucchini lethal chlorosis virus (ZLCV), gb: AOZ65578.1; Melon yellow spot virus (MYSV), gb: BAG70897; Gloxinia tospovirus, gb: AAC15466.1; Iris yellow spot virus (IYSV), gb: ACJ04669.1; Soybean vein necrosis virus (SVNV), gb: ADX96063.1; Calla lily chlorotic spot virus (CLCSV), gb: ACO52398.1; Capsicum chlorosis virus, gb: ABB83821.1; Polygonum ringspot tospovirus (PolRSV), gb: AHZ45964.1; Tomato zonate spot virus (TZSV), gb: YP_001740046.1; Chrysanthemum stem necrosis virus (CSNV), gb: BAF62146.1. Blue asterisks represent conserved cysteines in the Gn stem region among these viruses.

Fig. S7.

Amino acid alignment of C terminus of the Gn ectodomain among Bunyaviridea family. SFTSV (Database sequence accession no.: gb: AFB82725.1) and RVFV (gb: YP_003848705.1) belong to Phlebovirus genus; Bunyamwera virus (gb: AAA42777.1) represents Orthobunyavirus genus; Crimean-Congo hemorrhagic fever nairovirus (CCHFV, gb: AAM48107.1) represents Nairovirus genus, PUUV (gb: CAB43026.1) represents Hantavirus genus, and Tomato spotted wilt virus (TSWV), gb: NP_619703.1 represents Tospovirus genus. Blue asterisks represent conserved cysteines in the Gn stem region among these viruses.

Human Antibody MAb4-5 Specificity to SFTSV Gn.

Antibody MAb 4–5 was previously isolated using whole SFTSV virions as bait from a phage-display antibody library derived from the peripheral blood mononuclear cells of a patient that recovered from SFTS disease (30). Since only the sequence of the variable region was available, we constructed the Fab fragment with the variable region of MAb 4–5 heavy and light chains, combined with the constant region of a hemagglutinin antibody CR8020 (IgG1) (33). To verify neutralizing activity, we also constructed full-length MAb 4–5 with human Ig G1 (IgG1). The recombinant antibodies were expressed by 293T mammalian cells and the concentration required to obtain 50% neutralization of 100 TCID₅₀ SFTSV in vitro was 44.2 μg/mL (Fig. 3A). The interaction between Gn and MAb 4–5 was further demonstrated by surface plasmon resonance (SPR) assays with a dissociation constant (K_d) of 25.9 nM (Fig. 3B). We also measured the binding between RVFV Gn and MAb 4–5, but no binding or neutralization was observed, indicating the specific binding of MAb 4–5 to SFTSV (Fig. 3 C and D).

Fig. 3.

Neutralization and binding of MAb 4–5 to SFTSV and RVFV. (A) Neutralization potency of MAb 4–5 to SFTSV. A human monoclonal antibody (13C6) against Ebola was used as a negative control. (B) An SPR assay characterizing the specific binding between the SFTSV Gn head and MAb 4–5, with fitting curves in dashed line. The same data were plot as Scatchard. (C) Neutralization potency of MAb 4–5 to RVFV, showing negative results. (D) An SPR assay characterizing the specific binding between RVFV Gn head and MAb 4–5, showing no binding.

Complex Structure of the SFTSV Gn Head and Neutralizing MAb 4–5.

To elucidate the structural basis of virus neutralization, we further prepared the Gn head–MAb 4–5 Fab complex by mixing the two proteins in vitro and then purifying by size-exclusion chromatography (Fig. S8). Consistent with the high binding affinity between Gn head and MAb 4–5, the complex is stable and easily obtained. Crystals diffracting to 2.1 Å were grown from digested Gn in complex with MAb 4–5 Fab (Table S1). The complex structure was determined by molecular replacement using a Fab structure (PDB ID code 4RIR) as the search model followed by iterative rounds of model building and refinement. Automatic model extension of the missing domain of the Gn head was carried out with AUTOBUILD in PHENIX (34). Therefore, the SFTSV Gn structure described earlier was actually solved by “antibody-walking.” Structure analysis reveals that MAb 4–5 binds the membrane-distal head of Gn and the contact was mediated only by the heavy chain (Fig. 4A). The interaction surface between MAb 4–5 and Gn buries 614.8 Ų of the molecular surface. The primary region of the interaction is the complementarity-determining region (CDR) H3 (Fig. 4B), which penetrates the hydrophobic trough, consisting of α5, α6, and η5 in SFTSV Gn domain III. In this interaction, Y104 of the CDR H3 interacts with F256, F286, V289, and A290. Notably, α6 is the key element on the Gn head domain, which contacts all three CDR regions in the MAb 4–5 heavy chain. In particular, K288 is the most important residue located on α6. Specifically, the K288 main chain hydrogen-bonds the W33 side chain on the CDR H1 loop. The side chain of K288 can form salt bridges with the side chains of both D55 and D57 on the CDR H2 loop. K288 main chain hydrogen bonds the side chain of R101 on the CDR H3 loop. Moreover, two electrostatic patches are observed on the antigen–antibody interface. The basic patches, which consist of K100 and R102 on the CDR H3 loop, interact with E293 on Gn, while the acidic patch (D55 and D57) on the CDR H2 loop forms salt bridges with Gn K288 (Fig. 4C). To determine the critical role of residue 288 in MAb 4–5 binding, two substitutions (K288A and K288E) were constructed, expressed, and purified. Affinity assay indicates that these two substitutions dramatically decrease the binding between Gn and MAb 4–5 (Fig. S9).

Fig. S8.

Size-exclusion chromatography of the Gn head (red), MAb 4–5 (black) and the Gn head/MAb 4–5 complex (blue) in SFTSV. The prepared samples were loaded onto a Superdex 200 column (10/300 GL) individually. The overlaid chromatographs are shown, with the SDS/PAGE profiles of the pooled samples presented in the figure.

Fig. 4.

Crystal structure of MAb 4–5 Fab in complex with the SFTSV Gn head. (A) Overall structure of SFTSV Gn head and neutralizing antibody MAb 4–5 complex. The Gn head is presented as a cartoon diagram with the color in agreement with Fig. 1, while MAb 4–5 is shown as a surface representation with heavy chain in wheat and light chain in light blue. The detailed interactions between Gn and MAb 4–5 are highlighted in the box. (B) Surface representation of the interface between the SFTSV Gn head (Upper) and MAb 4–5 (Lower). CDR H1 is colored chartreuse; CDR H2, yellow; CDR H3, hotpink. The footprint on SFTSV Gn is colored according to the CDR that mediates the contact. Gn residues contacted by MAb 4–5 are indicated and colored accordingly. (C) The surface of the Gn head and MAb 4–5 colored for electrostatic potential: blue (basic), white (neutral), and red (acidic) at ±60 kTe⁻¹.

Fig. S9.

MAb 4–5 binding analysis of Gn wild-type and mutants: (A) Wild-type Gn, (B) Gn-K288A, (C) Gn-K288E.

The MAb 4–5 can recognize the denatured SFTSV Gn head but not the RVFV Gn head by Western blot (Fig. 5A). Structural analysis indicates that α6 is the major epitope recognized by MAb 4–5. Comparison of the epitope amino acids sequence between SFTSV and RVFV shows that only two amino acids are conserved (F286 and C287, SFTSV numbering) (Fig. 5B). The key residue K288 is replaced by serine in RVFV, which is the main reason leading the abolishment of binding to MAb 4–5. Specifically, the salt bridges between K288 and D55, K288 and D57 were lost. The hydrogen bonds between the Gn head and heavy chain were lost due to the shift of the main chain (Fig. 5C). Moreover, the hydrophobic pocket in SFTSV Gn can accommodate the side chain of F104 in the MAb 4–5 heavy chain. In contrast, the pocket becomes shallow (Fig. 5D), as A290 is replaced by Y423 in RVFV. Additionally, the side chain of K405 displays steric hindrance to CDR H3 loop. On the other hand, K405 in RVFV is a positive-charged amino acid, which shows repulsive force to the positive-charged R102 on the CDR H3 loop. Further conservation analysis of surface amino acids among 11 phleboviruses indicates that domain III is a variable region, which can be recognized by a specific neutralizing antibody (Fig. S10A). In contrast, domain II shows the relatively conserved epitope, which may be recognized by broadly neutralizing antibody (Fig. S10 B and C).

Fig. 5.

The epitope recognized by MAb 4–5. (A) Western blot analysis of the purified SFTSV Gn head and RVFV Gn head using MAb 4–5, showing the SFTSV Gn head can be detected by MAb 4–5 (with molecular mass of 37 kDa), while the RVFV Gn head cannot be detected. (B) Alignment of the epitope amino acid sequences between SFTSV and RVFV. (C) Superposition of the epitope structures between SFTSV (green) and RVFV (limon). MAb 4–5 is shown in wheat. (D) Comparison of the interaction details within hydrophobic pocket between SFTSV and RVFV Gn. The pocket is in surface representation and its contacting entities are in cartoon mode. Residues are labeled. The colors are consistent with C. K405 in the RVFV Gn and R102 in the Mab 4–5 CDR H3 loop are displayed in blue and marine surfaces.

Fig. S10.

Gn sequence conservation mapped onto the protein surface. The Gn surface is colored using the ConSurf server (62, 63) according to sequence conservation from the most divergent (dark cyan) to the most conserved (dark magenta). Eleven phleboviruses used for analysis are the same as shown in Fig. S3A. Domains I, II, and III are labeled accordingly. MAb 4–5 are indicated in cartoon with the color in agreement with Fig. 4. (A) The front view of the Gn sequence conservation map. (B) A clockwise rotation of A along a longitudinal axis. (C) A clockwise rotation of B along a longitudinal axis.

Discussion

In this study, we have reported two Gn head structures, each with a distinct architecture. Interestingly, a recent Gn structure from PUUV (32) is topologically different from the structures reported here. This may imply that viruses from different genera in the Bunyaviridae family may have different virus envelope protein architecture, which would be an interesting topic to be studied in the future.

So far, the real pattern of the arrangement of viral glycoproteins on the virus surface (how Gn and Gc, and other members if any, assemble into a functional complex) has not been determined clearly for any members of the bunyaviruses, even though a study proposing a low-resolution model was recently published (32). Previous studies have shown the Gn and Gc in several members of the phleboviruses—such as RVFV (35), UU.K.V (36), and Punta Toro virus (PTV) (37)—can be isolated as a heterodimer from mature virions or the infected cells. The Gn/Gc heterodimer may further assemble to form higher-ordered assemblies or “spikes” on the virion surface for proper transit to the Golgi apparatus (38), and a tetrameric model was proposed in the recent study of PUUV (32). However, Gn homodimers have also been isolated from UU.K.V virions (39), indicating that this kind of assembly cannot be ruled out. Moreover, disulfide-linked viral glycoprotein dimers were also reported in other related viruses: for example, measles virus hemagglutinin in measles virus (40–42) and hemagglutinin-esterase protein in the subset of the betacoronaviruses (43). This raises the possibility that dimerization of viral surface proteins plays an important role in attachment and entry into the host cell. Based on the biochemical assays, we propose here that the Gn dimer linked by the C-terminus disulfide bonds is likely the basic unit on the virus surface, and further assembles to form higher-ordered organization (Fig. 6A). To prove our model, the crystal structure of RVFV Gn was fitted into the T = 12 icosahedral cryo-EM map of RVFV (44) (Fig. 6B). There are five Gn molecules in each of 12 five-coordinated capsomers and six Gn molecules in each of 110 six-coordinated capsomers in the glycoprotein shell of RVFV (Fig. 6 B and C). Each pair of closest Gn molecules from two neighboring capsomers form a Gn dimer (Fig. 6 C and D). There are 360 Gn dimers in one RVFV particle in total. Moreover, the cryo-EM structure-fitting data of both RVFV and PUUV show that Gc occupies the inner half of the glycoprotein shell, while Gn is exposed outside, indicating that Gn is the major target for antibodies after infection. Additionally, it is clear that the structure of all solved Gc display typical characteristics of a class II fusion protein (24, 25, 45, 46), and the Gc have played a key role in fusion process. Gn likely contributes to receptor binding according to its position on the viral surface and our work reported here. However, determining the real function of the SFTSV Gn protein and the organization of two envelope proteins (Gn and Gc) within the virions will require high-resolution cryo-EM data and the structural characterization of Gn/Gc complex.

Fig. 6.

Proposed organization of the Gn on viral surface. (A) The anchoring model of Gn in phleboviruses. The Gn head domains are shown with the color in agreement with Fig. 1. The stem region is displayed with rounded rectangles in dark gray. The 10 cysteines in the stem region are displayed, and six paired cysteines are labeled with the same color (green, yellow, blue, orange, pink, and magenta). The cysteines responsible for dimerization are labeled in white. The transmembrane topology of Gn is indicated with green cylinders. The viral envelope is displayed with a modeled lipid-bilayer membrane. (B) Gn dimers in the glycoprotein shell of RVFV. The crystal structure of RVFV Gn was fitted into the capsomers in the cyro-EM map of RVFV (EMDataBank ID code EMD-1550). Five Gn molecules are in each of 12 five-coordinated capsomers and six Gn molecules in each of 110 six-coordinated capsomers. Each pair of the closest Gn molecules from two neighboring capsomers form a Gn dimer shown in the same color. One asymmetric unit of the particle is labeled in triangle shape (orange). (C) One of the 20 triangular faces of the icosahedral shell of RVFV, containing three pentons and seven hexons. (D) The side view of the Gn dimer between two capsomers in a cyro-EM map. The Gn dimers are shown in red cartoon and the EM map is displayed in gray surface.

Another interesting question is whether the MAb 4–5 has broad neutralizing capacity for the members of the Phlebovirus genus in Bunyaviridae family. Members in the Phlebovirus genus can initially be classified into two groups: the Sandfly fever group and the Uukuniemi group based on the antigenic, genomic, and vector relationships (47). RVFV belongs to the Sandfly fever group. Phylogenetic studies indicate that SFTSV represents a third distinct group within the Phlebovirus genus, which is transmitted by ticks (2). Furthermore, another phlebovirus, Heartland virus (HRTV), which was isolated from humans during 2012 in the United States, is closely related to SFTSV (48), with 61.9% amino acid identity in the M segment. K288 (SFTSV numbering) of SFTSV Gn is the key residue involved in MAb 4–5 binding. The corresponding residue in HRTV is arginine, which is also a positively charged amino acid, suggesting HRTV Gn may be capable of binding to MAb 4–5, and being neutralized. For other phleboviruses, the corresponding residues are negative-charged amino acids (glutamine) or uncharged hydrophilic amino acids (serine, threonine, and asparagine), which may not be neutralized by MAb 4–5. Moreover, E293 (SFTSV numbering) is another important residue in MAb 4–5 binding, which has electrostatic interaction with a positive-charged group (K100, R101, and R102) in the CDR H3 loop. However, not all of the residues in other phleboviruses are negative-charged amino acids. Take RVFV as an example: the corresponding residue is lysine (K), which has electrostatic repulsion to the positive-charged group in CDR H3 loop.

Although no specific host cell receptors have been identified in this genus thus far, the complex structure of Gn with its neutralizing antibody MAb 4–5 implies that the receptor binding site is likely located around the α6 helix in SFTSV. Alignment of the α6 helix in phleboviruses indicates that only C287 is conserved. Moreover, cell tropism among these viruses are not identical as well, suggesting their differences in receptor requirements. Specifically, RVFV can infect the thymus, spleen, and liver. FACS analysis in RVFV-GFP–infected mice showed that the macrophages, DCs, and granuclocytes were the main target cells for RVFV (49). SFTSV Gn/Gc pseudogtypes infected human lung (BEAS-2B, A549 and H1299), kidney (293T), liver (HepG2), colon (Caco-2), retinal epithelium (RPE), and glioblastroma (U373) cell lines, as well as human monocyte-derived DCs. Monocytic (THP-1) cells HFF and cervical carcinoma (HeLa) cells were resistant to SFTSV (27).

Our work represents structures of Gn proteins in the Phlebovirus genus of the Bunyaviridae family, and provides a detailed view of the interaction between SFTSV Gn and a neutralizing antibody. The Gn in RVFV and SFTSV display a novel fold, and four cysteines in the C terminus play important roles in dimerization among members in the whole Bunyaviridae. Moreover, the complex structure provides important information for the immune epitope, which may have implications for design of vaccines that are capable of eliciting effective immune responses against phleboviruses.

Materials and Methods

Protein Expression and Purification.

The SFTSV Gn ectodomain (GenBank accession no. JF906057.1, residues 20–452) followed by a C-terminal six-histidine purification tag was subcloned into the pFastBac1 vector (Invitrogen) modified with a gp67 signal sequence at the N terminus, as previously described (50–53). Transfection and virus amplification were conducted with sf9 cells, and the recombinant proteins were produced in High Five cells. The cell culture media were collected 60 h after infection and then purified by nickel affinity chromatography with a 5-mL HisTrap HP column (GE Healthcare) and size-exclusion chromatography with a Hiload 16/60Superdex 200-pg column in 20 mM Tris, pH 8.0, 50 mM NaCl. Gn protein was pooled and incubated with trypsin at a mass ratio of 300:1 at 277 K overnight, and further purified on a Superdex 200 column (GE Healthcare). Digested Gn was then concentrated to 7.5 mg mL⁻¹ for crystallization. The RVFV Gn head domain (GenBank accession no. JQ068143.1, residues 154–469) was constructed using the same strategy without trypsin digestion. The Gn head was concentrated to 10 mg mL⁻¹ for crystallization. To prove the C terminus of Gn playing an important role in dimerization, the Gn-C (residues 338–452) was constructed using the same strategy as the SFTSV Gn ectodomain described above. Moreover, the expression and purification strategies of Gn-C were the same as SFTSV Gn as well.

MAb 4–5 Fab was synthesized with its variable region (30) and the constant region of CR8020 IgG1 (33) into the pcDNA4 expression vector. A six-histidine tag was designed at the C terminus of the light chain for purification. The MAb 4–5 heavy chain was subcloned into a modified pcDNA4 with mouse IgG Fc at the C terminus. The full-length and the Fab fragment of MAb 4–5 were produced by HEK293T cells and purified by protein A and HisTrap HP column, respectively. MAb 4–5 Fab was further purified on a Superdex 200 column in 20 mM Tris, pH 8.0, 50 mM NaCl for crystallization, while the full-length MAb 4–5 was purified on a Superdex 200 column in PBS buffer for neutralization assay.

To obtain the complex of Gn and MAb 4–5, MAb 4–5 was mixed with the digested Gn at a molar ratio of 1:1 and incubated at 277 K for 1 h. The mixture was then loaded on a Superdex 200 column in 20 mM Tris, pH 8.0, 50 mM NaCl, and concentrated to 10 mg mL⁻¹ for crystallization.

Crystallization.

All crystallizations were performed using a vapor-diffusion sitting-drop method with 1 μL protein mixing with 1 μL reservoir solution. SFTSV Gn crystals were grown in the reservoir solution comprising of 20% (wt/vol) PEG 4000, 20% (vol/vol) 2-propanol, and 0.1 M sodium citrate, pH 5.5, at 291 K. RVFV Gn head crystals were grown in the reservoir solution of 0.2 M ammonium sulfate, 0.1 M Mes, pH 6.5, 20% (wt/vol) PEG 8000 at 277 K. Good diffraction crystals of the complex protein were finally obtained in 0.3 M potassium/sodium tartrate, 20% (wt/vol) PEG 3350, and 0.1 M Bis Tris propane, pH 7.5, with protein concentration of 15 mg mL⁻¹. Crystals were frozen in liquid nitrogen in reservoir solution supplemented with 20% glycerol (vol/vol) as a cryoprotectant. Data were collected at the Shanghai Synchrotron Radiation Facility BL17U. All data were processed with HKL2000.

Structure Determination and Refinement.

The structure of the SFTSV Gn head-MAb 4–5 complex was determined by molecular replacement using an antibody Fab fragment (PDB ID code 4RIR) as search probe. The molecular model was rebuilt using COOT (54) and refined with REFMAC (55). Subsequently, the automatic model extension of the missing Gn domain was carried out with AUTOBUILD in PHENIX (34). The method used for determination of the unknown structure by getting the phasing information from antibody is called “antibody walking.” The SFTSV Gn head structure was solved by molecular replacement using the refined coordinates obtained from the complex and the structure was refined using REFMAC. The RVFV Gn head structure was solved by single-wavelength anomalous diffraction, with a gold derivative (NaAuCl₄·2H₂O). Glycans were added at the final stages of model building and refinement according to the density map. Structure validation was performed with PROCHECK (56). Data collection and refinement statistics are summarized in Table S1. Figures were prepared with PyMOL (www.pymol.org).

Binding Assays.

Protein interactions were tested using both SPR analysis and the Octet RED96 biosensor method. For the SPR assays, a BIAcore 3000 spectrometer was used to measure kinetic constants at room temperature (298 K). All proteins were exchanged into a buffer of 20 mM Hepes, pH 7.4, 150 mM NaCl and 0.005% (vol/vol) Tween 20. MAb 4–5 proteins were immobilized on CM5 chips (GE Healthcare) at approximately 1,000 response units and analyzed for real-time binding by flowing through gradient concentrations (ranging from 7.8 to 1,000 nM) of Gn head proteins. For the Octet RED96 biosensor method, samples of buffer were dispensed into polypropylene 96-well black flat-bottom plates (Greiner Bio-One) at a volume of 200 μL per well, and all measurements were performed at 298 K in HBS-EP buffer with the plate shaking at the speed of 1,000 rpm. Anti-human IgG Fc (AMC)-coated biosensor tips (Pall ForteBio) were used to capture antibody MAb 4–5 from 10 ng/μL stock buffer. The buffer was 20 mM Hepes, pH 7.4, 150 mM NaCl and 0.005% (vol/vol) Tween 20. Kinetic measurements for antigen binding were performed by exposing biosensors to a series of analyte concentrations (15.6−250 nM for Gn wild-type, and 15.6–500 nM for Gn mutants) and background subtraction was used to correct for sensor drifting. All sensors were generated with 10 mM Glycine-HCl, (pH 1.7, GE Healthcare). The data were processed by ForteBio’s data analysis software and plotted by Origin software.

Neutralization Assay.

Fifty-microliters of twofold serial-diluted MAb 4–5 or control antibody (Ebola monoclonal antibody 13C6) (57) was mixed with equal volume of 100 TCID₅₀ SFTSV (SDYY007), or RVFV (BJ01) at 310 K for 1 h. The virus–antibody mixture was then transferred to 80% confluent Vero cells in a 96-well plate and incubated at 310 K for 1 h. The plate was washed with DMEM three times, and incubated at 310 K in a 5% CO₂ incubator. Cytopathic effects were observed every 24 h for 6 d. The antibody was considered as having neutralizing capacity if 100% of viral cytopathic effects was inhibited.

Identification of Protein Disulfide Bonds by MS Analysis.

Purified SFTSV Gn-C was denatured in a buffer containing 8 M Urea, 1 mM Tris, pH 6.5, 2 mM N-ethylmaleimide before it was subjected to SDS/PAGE. The monomer and dimer bands of Gn-C were excised and digested in gel according to a previously published protocol (58), with the following modifications: (i) reduction and alkylation were omitted, (ii) 0.5 mM N-ethylmaleimide was added to all of the destaining solutions and wash buffers, and (iii) dehydrated gel slices were rehydrated with 100 mM Tris, pH 6.5, containing Lys-C and Asp-N at 5 ng/µL each for overnight digestion. Digested peptides were analyzed on a Q Exactive mass spectrometer coupled to an Easy Nano-LC 1000 liquid chromatography system (Thermo Fisher Scientific).

Peptides were desalted on a 75-μm × 6-cm precolumn that was packed with 10 μm, 120 Å ODS-AQ C18 resin (YMC Co., Ltd.) and connected to a 75-μm × 10-cm analytical column packed with 1.8 μm, 120 Å UHPLC-XB-C18 resin (Welch Materials). The peptides were separated over a 34-min linear gradient from 5% buffer B (100% acetonitrile, 0.1% formic acid), 95% buffer A (0.1% formic acid) to 30% buffer B, followed by a 3-min gradient from 30 to 80% buffer B, then maintaining at 80% buffer B for 7 min. The flow rate was 200 nL/min. The MS parameters were as follows: the top 20 most-intense ions were selected for HCD dissociation; R = 140,000 in full scan, R = 17,500 in HCD scan; AGC targets were 1e6 for FTMS full scan, 5e4 for MS2; minimal signal threshold for MS2 = 4e4; precursors having a charge state of +1, >+8, or unassigned were excluded; normalized collision energy, 30; peptide match, preferred.

The raw data of the 10-protein sample were preprocessed using pParse (59), which was set to exclude coeluting precursor ions and precursors of +1, +2 charge. To identify disulfide-linked peptides, the MS2spectra was searched using pLink (60) against a protein database containing the sequences of the analyte protein and all of the proteases used. The pLink parameters search were maximum number of missed cleavages = 5; minimum peptide length = 4 amino acids; fixed modification of −1.007285 Da on cysteine and the disulfide mass was set to zero to allow the identification of more than one disulfide bond in a peptide pair; candidate pairs satisfying Mα+ Mβ+ Mlinker < M ± 5 Da were scored for spectrum-peptide matching (Mα, Mβ, Mlinker, and M denote the masses of the candidate α-peptide, candidate β-peptide, the linker, and the observed precursor, respectively). pLink search results were filtered by requiring E-value < 0.001, false-discovery rate < 0.05, spectra count ≥ 20, and no more than 10-ppm deviation between the monoisotopic masses of the observed precursor and the matched disulfide-linked peptide (or peptide pair).

Western Blot Analysis.

The SFTSV and RVFV Gn head proteins were fractionated by 12% SDS/PAGE. The separate proteins were electro-transferred to a nitrocellulose membrane and incubated with full-length MAb 4–5 antibody and a horseradish peroxidase-conjugated secondary goat anti-human IgG antibody (SC-2453; Santa Cruz). SuperSignal West Pico Chemiluminescent Substrate (Pierce 34080) was used for detection according to the manufacturer’s instructions.

Fitting of the RVFV Gn Structure into the Low-Resolution EM Structure.

The crystal structure of RVFV Gn was manually fitted into the capsomers in the 22-Å resolution cyro-EM map of RVFV (EMDataBank ID code EMD-1550) with UCSF Chimera (61). The positions of Gn with the five- and six-coordinated capsomers were adjusted according to the fivefold and sixfold rotational symmetry, respectively, without major steric clashes.