Structures of phlebovirus glycoprotein Gn andidentification of a neutralizing antibody epitopeYan Wua,b,1, Yaohua Zhuc,d,1, Feng Gaoe,1,2, Yongjun Jiaof,1, Babayemi O. Oladejoa, Yan Chaia, Yuhai Bia,b,g, Shan Luh,Mengqiu Dongh, Chang Zhanga, Guangmei Huanga, Gary Wonga, Na Lii, Yanfang Zhanga, Yan Lia, Wen-hai Fengc,d,Yi Shia,b,g, Mifang Liangj, Rongguang Zhangi, Jianxun Qia, and George F. Gaoa,b,g,i,j,k,2

aChinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing100101, China; bShenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, Shenzhen 518112, China; cState Key Laboratory ofAgrobiotechnology, Beijing 100193, China; dDepartment of Microbiology and Immunology, College of Biological Sciences, China Agricultural University,Beijing 100193, China; eInstitute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; fInstitute of PathogenicMicrobiology, Jiangsu Provincial Center for Disease Prevention and Control, Key Laboratory of Enteric Pathogenic Microbiology, Ministry Health, Nanjing210009, China; gCenter for Influenza Research and Early-Warning, Chinese Academy of Sciences, Beijing 100101, China; hNational Institute of BiologicalSciences, Beijing 102206, China; iNational Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for BiologicalSciences, Chinese Academy of Sciences, Shanghai 201210, China; jNational Institute for Viral Disease Control and Prevention, Chinese Center for DiseaseControl and Prevention, Beijing 102206, China; and kResearch Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academyof Sciences, Beijing 100101, China

Edited by Stephen C. Harrison, Children’s Hospital Harvard Medical School and Howard Hughes Medical Institute, Boston, MA, and approved July 25, 2017(received for review March 30, 2017)

Severe fever with thrombocytopenia syndrome virus (SFTSV) andRift Valley fever virus (RVFV) are two arthropod-borne phlebovi-ruses in the Bunyaviridae family, which cause severe illness inhumans and animals. Glycoprotein N (Gn) is one of the envelopeproteins on the virus surface and is a major antigenic component.Despite its importance for virus entry and fusion, the molecularfeatures of the phleboviruse Gn were unknown. Here, we presentthe crystal structures of the Gn head domain from both SFTSV andRVFV, which display a similar compact triangular shape overall,while the three subdomains (domains I, II, and III) making up theGn head display different arrangements. Ten cysteines in the Gnstem region are conserved among phleboviruses, four of which areresponsible for Gn dimerization, as revealed in this study, and theyare highly conserved for all members in Bunyaviridae. Therefore,we propose an anchoring mode on the viral surface. The complexstructure of the SFTSV Gn head and human neutralizing antibodyMAb 4–5 reveals that helices α6 in subdomain III is the key com-ponent for neutralization. Importantly, the structure indicates thatdomain III is an ideal region recognized by specific neutralizingantibodies, while domain II is probably recognized by broadly neu-tralizing antibodies. Collectively, Gn is a desirable vaccine target,and our data provide a molecular basis for the rational design ofvaccines against the diseases caused by phleboviruses and a modelfor bunyavirus Gn embedding on the viral surface.

bunyavirus | SFTSV | glycoprotein | neutralizing antibody | RVFV

The Bunyaviridae is a large family of human, animal, and plantpathogens spanning five genera: Orthobunyavirus, Hantavirus,

Nairovirus, Phlebovirus, and Tospovirus (1). With the exception ofhantaviruses, all other bunyaviruses are arthropod-borne viruses.Severe fever with thrombocytopenia syndrome virus (SFTSV) andRift Valley fever virus (RVFV) belong to the Phlebovirus genus,which can cause emerging infectious diseases in humans (1–3), asemphasized by the recent imported case of RVFV infection in Chinaafter 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 presentedwith fever, thrombocytopenia, leukocytopenia, and multiorgandysfunction until 2011 (2, 5). SFTSV-infected patients have beenfound in at least 13 provinces in China, with a case fatality rate of≈12%. In 2013, cases of SFTS were reported in South Korea andJapan, with a case fatality rate of 35.4% and 50%, respectively(6–8). Unfortunately, vaccine or antiviral intervention againstSFTSV 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 out-breaks of RVFV disease have been reported in ruminants andhumans in Africa and the Arabian Peninsula (11, 12), consti-tuting a significant threat to global public health and agriculture.Humans can be infected by bites from virus-carrying mosquitoesor through contact with bodily fluids of the infected animals (13).RVF patients display a self-limiting febrile illness, and somecases may develop lethal hemorrhagic fever, neurologic disor-ders, or blindness (13). Vaccines have been used to control RVFamong livestock in endemic regions in Africa and the ArabianPeninsula (14, 15). However, formalin-inactivated whole-virus vac-cines show little immunogenicity (16, 17), whereas live-attenuatedvaccine is teratogenic in pregnant sheep and cattle (18, 19). Cur-rently, there are no effective vaccines or antiviral agents approvedfor use in humans.

Significance

Bunyaviruses are emerging zoonotic pathogens of public-health concern. Lack of structures for proteins on the viralmembrane (“envelope”) surface limits understanding of entry.We describe atomic-level structures for the globular “head” ofthe 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 thebunyavirus family, and a structure of the SFTSV Gn bound witha neutralizing antibody Fab. The results show the folded Gnstructure and define virus-specific neutralizing-antibody bind-ing sites. Biochemical assays suggest that dimerization, medi-ated by conserved cysteines in the region (“stem”) connectingthe Gn head with the transmembrane domain, is a generalfeature of bunyavirus envelope proteins and that the dimer isprobably the olimeric form on the viral surface.

Author contributions: Y.W. and G.F.G. designed research; Y.W., Y. Zhu, F.G., B.O.O., Y.B.,S.L., C.Z., G.H., Y. Zhang, Y.L., and J.Q. performed research; Y.J., W.F., M.L., and R.Z.contributed new reagents/analytic tools; Y.W., F.G., Y.C., S.L., M.D., N.L., Y.S., andG.F.G. analyzed data; and Y.W., F.G., G.W., and G.F.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.wwpdb.org (PDB ID codes: 5Y0W, 5Y0Y, 5Y10, and 5Y11).1Y.W., Y. Zhu, F.G., and Y.J. contributed equally to this work.2To whom correspondence may be addressed. Email: gaofeng@genetics.ac.cn or gaof@im.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1705176114/-/DCSupplemental.

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As with the other members of the Bunyaviridae family, SFTSVand RVFV genomes contain three negative-stranded RNA seg-ments (L, M, and S) (1). The M segment encodes a glycoproteinprecursor, which can be cleaved by cellular proteases duringtranslation (20). For SFTSV, the precursor can be processedinto two subunits: glycoprotein N (Gn) and glycoprotein C (Gc)(1), while for RVFV, one more subunit, called nonstructuralprotein 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 solvedcrystal structures of RVFV and SFTSV Gc proteins revealedarchitectural similarity with class II viral fusion proteins (24, 25).Low pH can induce RVFV Gc oligomerization, but has no in-fluence on Gn (23). Recent studies reveal that the lectin dendritic-cell (DC) specific intercellular adhesion molecule 3-grabbingnonintegrin (SIGN) is identified as the entry factor required formany phleboviruses, including SFTSV, RVFV, Toscana virus (TOSV),and Uukuniemi virus (UU.K.V) (26, 27). L-SIGN, another C-typelectin, shares 77% sequence homology with DC-SIGN and acts asan attachment receptor for these phleboviruses, rather than as anendocytic receptor (28). Nonmuscle myosin heavy chain II A(NMMHC-IIA) is reported as a critical factor contributing to theefficiency of early SFTSV infection, and the recombinant Gnprotein is capable of binding NMMHC-IIA, indicating that Gn islikely 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 an-tibody targeting Gn, showing cross-neutralizing activity to a widerange of SFTSV isolates in China (30). An RVFV Gn/Gc subunitvaccine was previously shown to elicit a strong neutralizing an-tibody 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 incomplex with MAb 4–5, a neutralizing antibody identified in SFTSrecovered patients (30). These two Gn head domains display asimilar overall configuration but a different overall topology to theGn head structure of Puumala hantavirus (PUUV) (32). Fourcysteine residues highly conserved in the stem region of Gn areresponsible for dimerization, suggesting the Gn cysteine-mediateddimer model might apply to the entire Bunyaviridae family. Thecomplex structure reveals that the key residues in SFTSV recog-nized by neutralizing MAb 4–5 is not conserved in RVFV; there-fore, MAb 4–5 cannot bind to RVFV Gn. Moreover, conservedexposure amino acid alignment of 11 members in Phlebovirus genusindicates that domain III of Gn provide epitopes for specificneutralizing antibody, while domain II is probably an ideal regionrecognized by a broadly neutralizing antibody. Altogether ourfindings have proved that Gn is a promising antigen for vaccinedevelopment and the crystal structures provide a molecular basisfor the rational design of vaccines and antiviral drugs.

ResultsOverall Structure of the Gn Head Domain: SFTSV and RVFV. BothSFTSV and RVFV Gn are type I transmembrane proteins withthe N-terminal ectodomain binding on the cell surface and aC-terminal transmembrane helix anchored on the virus mem-brane (Fig. 1A). The ectodomain can be divided into the headand stem domains. To facilitate crystallization, the stem regionof SFTSV Gn was removed by limited trypsin digestion due to anunsuccessful effort with the full-length ectodomain for crystalli-zation. The last amino acid visible in the crystal structure isN340. However, the molecular weight measured by mass spec-trometry (MS) indicates that the cleavage site is between resi-dues K371 and S372, suggesting a conformational disorder in theC terminus of the truncated Gn (residues 340–372), instead ofdegradation during crystallization (Fig. 1A). We designed theconstruct of the RVFV Gn head domain based on the structure

of the SFTSV Gn head domain, and succeeded in obtaining itscrystal structure. Both the crystal structures of SFTSV and RVFVGn head domains were determined at a resolution of 2.6 Å (TableS1). A Dali search within the Protein Data Bank (PDB) failed toidentify any existing structures to the Gn of both SFTSV andRVFV, suggesting a novel fold. The structure of SFTSV Gn wassolved by “antibody-walking” (Materials and Methods), in which theantibody provides model-based phasing information to determinethe unknown structure. The RVFV Gn structure was solved bymolecular replacement, using SFTSV Gn as a search model.The Gn head domains of both SFTSV and RVFV fold into a

similar triangular shape architecture consisting of three sub-domains (Fig. 1 B and C). Subdomains I and II form the foun-dation bed supporting the subdomain III protruding on the top(Fig. 1 B and C and Fig. S1A). However, the three subdomainsshow a different arrangement between these two viruses, with anaverage rmsd of 5.010, 2.393, and 1.825 for subdomains I, II, andIII, respectively (Fig. S1B). Specifically, in subdomain I ofSFTSV, a five-stranded β-sheet (β2, β3, β4, β5, and β9), one helix(α3), and one 310-helix (η2) are located on the interface ofsubdomains 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. Afree cysteine (C99) on α3 is located in the interior of subdomainI. 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 foundon the interface of subdomains I and II. One α-helix (α1), three310-helices (η1, η2, and η3), and one pair of small antiparallelβ-strand (β4 and β5) are on the opposite side. β-Strands are themajor component in subdomain II and the pattern of β-strandsbetween SFTSV and RVFV is the same (Fig. 1 D and E and Fig.S2). The core structure comprising the six β-strands is locatednext to subdomain I and a three-stranded β-sheet connects tosubdomain III. The only different secondary element in sub-domain II between these two is the α-helix. SFTSV contains anα4, whereas RVFV does not have it in subdomain II. The sec-ondary elements in subdomain III between SFTSV and RVFVare similar. Four β-strands and three α-helices stabilize sub-domain III, in which α5 in SFTSV is replaced by η5 in RVFV(Fig. 1 D and E). Although the Gn structures in the Phlebovirusgenus display similar configurations, the overall structures be-tween the Phlebovirus genus and Hantavirus genus are distinct(Fig. S1 C and D). Furthermore, the topology between these twogenera is completely different (Fig. S1E).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, andD and Fig. S2), which is consistent with theoretical predictions.For RVFV, although N438 is predicted to be an N-linked gly-cosylation site, no glycans can be observed in the solved crystalstructure 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 thesetwo Gn, and all of the other 10 cysteines in the stem domains areconserved for the 11 sequences of the available phleboviruses (Figs.S2 and S3A). The SFTSV Gn head domain contains eight disulfidebonds and a free cysteine. Specifically, two disulfide bonds (C26-C49 and C143-C156) and an unpaired cysteine (C99) are in sub-domain I, one disulfide bond is in subdomain II (C206-C216), onedisulfide bond is across subdomains II and III (C180-C327), andfour disulfide bonds are in subdomain III (C258-C305, C266-303,C274-C280, and C287-C292). The RVFV Gn head domain is sta-bilized by nine disulfide bonds, four of which are in subdomain I(C179-C188, C229-C239, C250-C281, and C271-C284), one ofwhich is in subdomain II (C322-C332), three of which are in sub-domain III (C374-C434, C402-C413, and C420-C425), and one ofwhich is across subdomains II and III (C304-C456). Six disulfide

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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 con-served between the SFTSV and RVFV Gn head domains (Fig. S2).

Dimerization of Gn Through Its Stem Region. The full-length SFTSVGn ectodomain was produced using the baculovirus expressionsystem. Results from size-exclusion chromatography using aSuperdex 200 10/300 GL column showed the peaks eluted at12.6 mL and 14.4 mL, corresponding to the dimer and monomer

in solution, respectively (Fig. 2A). Nonreducing SDS/PAGEshows that the dimer is linked by disulfide bonds (Fig. S4A).Notably, the dimer was completely disassociated after a treat-ment of limited trypsin proteolysis (Fig. 2A and Fig. S4A),indicating that the C terminus of Gn is solely responsible fordimerization via disulfide bonds (Fig. 2A). To characterize theC terminus of Gn, we constructed the unstructured part of Gn(Gn-C), which corresponds to residues 338–452. The Gn-C proteinforms a dimer under nonreducing conditions, which is consis-tent with the above-described result (Fig. S4B). Full-length

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, subdomainI 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 thestem 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 IIIis 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 stemregion) 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 fol-lowing the same coloring scheme as in the cartoon representation, with detailed secondary structure elements and disulfide bonds (dashed lines labeled withSS). 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 cartoonrepresentation, with detailed secondary structure elements and disulfide bonds (dashed lines labeled with SS).

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RVFV Gn forms a dimer under nonreducing conditions as well(Fig. S4C).To determine which cysteines are responsible for dimerization,

we used MS and site-directed mutagenesis to analyze Gn-C andthe full-length Gn protein of SFTSV. MS analysis identified fourintermolecular disulfide bonds involving C430, C435, C438, andC447 from the Gn-C dimer but not the monomer (Table S2),suggesting that these cysteine residues may mediate Gn di-merization. Indeed, simultaneously mutating these four cysteineresidues to alanine completely abolished dimer formation of thefull-length Gn, leaving only the monomer (Fig. 2B). Further-more, the C430A/C447A substitution shifted the equilibriumtoward the Gn monomer, but failed to abolish the Gn dimer. TheC435A/C438A substitution reduced the relative abundance ofthe dimer more substantially than the C430A/C447A substitu-tion (Fig. 2B), suggesting that although C430, C435, C438, andC447 all contribute to Gn dimerization, the two intermoleculardisulfide bonds mediated by C435 and C438 play a major role.MS analysis also identified a disulfide bond between C356 andC424 in both the Gn-C monomer and dimer (Table S2). Muta-tion of either C356 or C424 severely reduced the yield of theprotein (Fig. S5), suggesting that the C356-C424 disulfide bond iscritical for stabilizing Gn. The data described above support thatthe cysteines in the stem region are responsible for dimerizationand 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 di-merization are highly conserved across five genera (Phlebovirus,Hantavirus, Nairovirus, Orthobunyavirus, and Tospovirus) of theBunyaviridae family, indicating that members in the wholeBunyaviridae likely share the same assembly organization with aGn disulfide bond-linked dimer (Fig. S7).

Human Antibody MAb4-5 Specificity to SFTSV Gn.Antibody MAb 4–5was previously isolated using whole SFTSV virions as bait from aphage-display antibody library derived from the peripheral bloodmononuclear 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 MAb4–5 heavy and light chains, combined with the constant region of a

hemagglutinin antibody CR8020 (IgG1) (33). To verify neutralizingactivity, we also constructed full-length MAb 4–5 with human IgG1 (IgG1). The recombinant antibodies were expressed by 293Tmammalian 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 furtherdemonstrated by surface plasmon resonance (SPR) assays with adissociation constant (Kd) of 25.9 nM (Fig. 3B). We also measuredthe binding between RVFV Gn and MAb 4–5, but no binding orneutralization was observed, indicating the specific binding of MAb4–5 to SFTSV (Fig. 3 C and D).

Complex Structure of the SFTSV Gn Head and Neutralizing MAb 4–5.To elucidate the structural basis of virus neutralization, we fur-ther prepared the Gn head–MAb 4–5 Fab complex by mixing thetwo proteins in vitro and then purifying by size-exclusion chro-matography (Fig. S8). Consistent with the high binding affinitybetween Gn head and MAb 4–5, the complex is stable and easilyobtained. Crystals diffracting to 2.1 Å were grown from digestedGn in complex with MAb 4–5 Fab (Table S1). The complexstructure was determined by molecular replacement using a Fabstructure (PDB ID code 4RIR) as the search model followed byiterative rounds of model building and refinement. Automaticmodel extension of the missing domain of the Gn head wascarried out with AUTOBUILD in PHENIX (34). Therefore, theSFTSV Gn structure described earlier was actually solved by“antibody-walking.” Structure analysis reveals that MAb 4–5binds the membrane-distal head of Gn and the contact wasmediated only by the heavy chain (Fig. 4A). The interactionsurface between MAb 4–5 and Gn buries 614.8 Å2 of the mo-lecular surface. The primary region of the interaction is thecomplementarity-determining region (CDR) H3 (Fig. 4B), whichpenetrates the hydrophobic trough, consisting of α5, α6, andη5 in SFTSV Gn domain III. In this interaction, Y104 of theCDR H3 interacts with F256, F286, V289, and A290. Notably,α6 is the key element on the Gn head domain, which contacts allthree 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 theCDR H1 loop. The side chain of K288 can form salt bridges with

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 trypsindigestion, indicating that the full-length of the Gn ectodomain has two states, monomer and dimer (black), while the Gn head is monomer only (blue andred). (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.

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the side chains of both D55 and D57 on the CDR H2 loop.K288 main chain hydrogen bonds the side chain of R101 on theCDR H3 loop. Moreover, two electrostatic patches are observedon the antigen–antibody interface. The basic patches, which consistof K100 and R102 on the CDRH3 loop, interact with E293 on Gn,while the acidic patch (D55 and D57) on the CDR H2 loop formssalt bridges with Gn K288 (Fig. 4C). To determine the critical roleof residue 288 in MAb 4–5 binding, two substitutions (K288A andK288E) were constructed, expressed, and purified. Affinity assayindicates that these two substitutions dramatically decrease thebinding between Gn and MAb 4–5 (Fig. S9).The MAb 4–5 can recognize the denatured SFTSV Gn head

but not the RVFV Gn head by Western blot (Fig. 5A). Structuralanalysis indicates that α6 is the major epitope recognized byMAb 4–5. Comparison of the epitope amino acids sequencebetween SFTSV and RVFV shows that only two amino acids areconserved (F286 and C287, SFTSV numbering) (Fig. 5B). Thekey residue K288 is replaced by serine in RVFV, which is themain reason leading the abolishment of binding to MAb 4–5.Specifically, the salt bridges between K288 and D55, K288 andD57 were lost. The hydrogen bonds between the Gn head andheavy chain were lost due to the shift of the main chain (Fig. 5C).Moreover, the hydrophobic pocket in SFTSV Gn can accom-modate the side chain of F104 in the MAb 4–5 heavy chain. Incontrast, the pocket becomes shallow (Fig. 5D), as A290 is

replaced by Y423 in RVFV. Additionally, the side chain ofK405 displays steric hindrance to CDR H3 loop. On the otherhand, K405 in RVFV is a positive-charged amino acid, whichshows repulsive force to the positive-charged R102 on the CDRH3 loop. Further conservation analysis of surface amino acidsamong 11 phleboviruses indicates that domain III is a variableregion, which can be recognized by a specific neutralizing anti-body (Fig. S10A). In contrast, domain II shows the relativelyconserved epitope, which may be recognized by broadly neu-tralizing antibody (Fig. S10 B and C).

DiscussionIn this study, we have reported two Gn head structures, each witha distinct architecture. Interestingly, a recent Gn structure fromPUUV (32) is topologically different from the structures reportedhere. This may imply that viruses from different genera in theBunyaviridae family may have different virus envelope proteinarchitecture, which would be an interesting topic to be studied inthe 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 determinedclearly for any members of the bunyaviruses, even though a studyproposing a low-resolution model was recently published (32).Previous studies have shown the Gn and Gc in several members of

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 fittingcurves 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 assaycharacterizing the specific binding between RVFV Gn head and MAb 4–5, showing no binding.

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the phleboviruses—such as RVFV (35), UU.K.V (36), and PuntaToro virus (PTV) (37)—can be isolated as a heterodimer frommature virions or the infected cells. The Gn/Gc heterodimer mayfurther assemble to form higher-ordered assemblies or “spikes” onthe 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 fromUU.K.V virions (39), indicating that this kind of assembly cannot be ruledout. Moreover, disulfide-linked viral glycoprotein dimers were alsoreported in other related viruses: for example, measles virus hem-agglutinin in measles virus (40–42) and hemagglutinin-esteraseprotein in the subset of the betacoronaviruses (43). This raises the

possibility that dimerization of viral surface proteins plays an im-portant role in attachment and entry into the host cell. Based on thebiochemical assays, we propose here that the Gn dimer linked by theC-terminus disulfide bonds is likely the basic unit on the virus sur-face, and further assembles to form higher-ordered organization(Fig. 6A). To prove our model, the crystal structure of RVFV Gnwas fitted into the T = 12 icosahedral cryo-EM map of RVFV (44)(Fig. 6B). There are five Gn molecules in each of 12 five-coordinatedcapsomers and six Gn molecules in each of 110 six-coordinatedcapsomers in the glycoprotein shell of RVFV (Fig. 6 B and C).Each pair of closest Gn molecules from two neighboring capsomersform a Gn dimer (Fig. 6 C and D). There are 360 Gn dimers in one

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 4–5complex. 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 withheavy 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 representationof 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 onSFTSV 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 surfaceof the Gn head and MAb 4–5 colored for electrostatic potential: blue (basic), white (neutral), and red (acidic) at ±60 kTe−1.

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RVFV particle in total. Moreover, the cryo-EM structure-fitting dataof both RVFV and PUUV show that Gc occupies the inner half ofthe glycoprotein shell, while Gn is exposed outside, indicating thatGn is the major target for antibodies after infection. Additionally, it isclear that the structure of all solved Gc display typical characteristicsof a class II fusion protein (24, 25, 45, 46), and the Gc have played akey role in fusion process. Gn likely contributes to receptor bindingaccording to its position on the viral surface and our work reportedhere. However, determining the real function of the SFTSV Gnprotein and the organization of two envelope proteins (Gn and Gc)within the virions will require high-resolution cryo-EM data and thestructural characterization of Gn/Gc complex.Another interesting question is whether the MAb 4–5 has

broad neutralizing capacity for the members of the Phlebovirusgenus in Bunyaviridae family. Members in the Phlebovirus genuscan initially be classified into two groups: the Sandfly fever groupand the Uukuniemi group based on the antigenic, genomic, andvector relationships (47). RVFV belongs to the Sandfly fevergroup. Phylogenetic studies indicate that SFTSV represents a thirddistinct group within the Phlebovirus genus, which is transmitted byticks (2). Furthermore, another phlebovirus, Heartland virus (HRTV),which was isolated from humans during 2012 in the United States, isclosely related to SFTSV (48), with 61.9% amino acid identity in theM segment. K288 (SFTSV numbering) of SFTSV Gn is the key res-idue 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 corre-sponding residues are negative-charged amino acids (glutamine)or uncharged hydrophilic amino acids (serine, threonine, andasparagine), which may not be neutralized by MAb 4–5. More-over, E293 (SFTSV numbering) is another important residue inMAb 4–5 binding, which has electrostatic interaction with apositive-charged group (K100, R101, and R102) in the CDRH3 loop. However, not all of the residues in other phlebovirusesare negative-charged amino acids. Take RVFV as an example: thecorresponding residue is lysine (K), which has electrostatic re-pulsion 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 neutralizingantibody MAb 4–5 implies that the receptor binding site is likelylocated around the α6 helix in SFTSV. Alignment of the α6 helix inphleboviruses indicates that only C287 is conserved. Moreover, celltropism among these viruses are not identical as well, suggestingtheir differences in receptor requirements. Specifically, RVFV caninfect the thymus, spleen, and liver. FACS analysis in RVFV-GFP–infected mice showed that the macrophages, DCs, and granuclocyteswere the main target cells for RVFV (49). SFTSVGn/Gc pseudogtypesinfected human lung (BEAS-2B, A549 and H1299), kidney (293T),liver (HepG2), colon (Caco-2), retinal epithelium (RPE), and

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 SFTSVGn 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 acidsequences 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 contactingentities 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 displayedin blue and marine surfaces.

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glioblastroma (U373) cell lines, as well as human monocyte-derivedDCs. 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 ofthe interaction between SFTSV Gn and a neutralizing antibody.The Gn in RVFV and SFTSV display a novel fold, and four cys-teines in the C terminus play important roles in dimerization amongmembers in the whole Bunyaviridae. Moreover, the complex struc-ture provides important information for the immune epitope, whichmay have implications for design of vaccines that are capable ofeliciting effective immune responses against phleboviruses.

Materials and MethodsProtein Expression and Purification. The SFTSV Gn ectodomain (GenBank ac-cession no. JF906057.1, residues 20–452) followed by a C-terminal six-histidinepurification tag was subcloned into the pFastBac1 vector (Invitrogen) modifiedwith 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 mediawere collected 60 h after infection and then purified by nickel affinity chro-matography with a 5-mL HisTrap HP column (GE Healthcare) and size-exclusionchromatography 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 ata mass ratio of 300:1 at 277 K overnight, and further purified on a Superdex200 column (GE Healthcare). Digested Gn was then concentrated to 7.5 mgmL−1

for crystallization. The RVFV Gn head domain (GenBank accession no.JQ068143.1, residues 154–469) was constructed using the same strategy with-out trypsin digestion. The Gn head was concentrated to 10 mg mL−1 for crys-tallization. To prove the C terminus of Gn playing an important role indimerization, the Gn-C (residues 338–452) was constructed using the samestrategy as the SFTSV Gn ectodomain described above. Moreover, the expres-sion 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 constantregion of CR8020 IgG1 (33) into the pcDNA4 expression vector. A six-histidinetag was designed at the C terminus of the light chain for purification. The MAb4–5 heavy chain was subcloned into a modified pcDNA4 with mouse IgG Fcat the C terminus. The full-length and the Fab fragment of MAb 4–5 wereproduced 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

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 inagreement 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 pairedcysteines 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 inthe 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 Gnmolecules 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 dimerbetween two capsomers in a cyro-EM map. The Gn dimers are shown in red cartoon and the EM map is displayed in gray surface.

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20 mM Tris, pH 8.0, 50 mMNaCl for crystallization, while the full-length MAb 4–5was 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 thedigested Gn at a molar ratio of 1:1 and incubated at 277 K for 1 h. Themixture 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-diffusionsitting-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 Mammonium sulfate, 0.1 M Mes, pH 6.5, 20% (wt/vol) PEG 8000 at 277 K. Gooddiffraction crystals of the complex protein were finally obtained in 0.3 M po-tassium/sodium tartrate, 20% (wt/vol) PEG 3350, and 0.1 M Bis Tris propane,pH 7.5, with protein concentration of 15 mgmL−1. Crystals were frozen in liquidnitrogen in reservoir solution supplemented with 20% glycerol (vol/vol) as acryoprotectant. Data were collected at the Shanghai Synchrotron RadiationFacility 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 an-tibody Fab fragment (PDB ID code 4RIR) as search probe. The molecularmodel was rebuilt using COOT (54) and refined with REFMAC (55). Sub-sequently, the automatic model extension of the missing Gn domain wascarried out with AUTOBUILD in PHENIX (34). The method used for de-termination of the unknown structure by getting the phasing informationfrom antibody is called “antibody walking.” The SFTSV Gn head structurewas solved by molecular replacement using the refined coordinatesobtained from the complex and the structure was refined using REFMAC.The RVFV Gn head structure was solved by single-wavelength anomalousdiffraction, with a gold derivative (NaAuCl4·2H2O). Glycans were added atthe final stages of model building and refinement according to the densitymap. Structure validation was performed with PROCHECK (56). Data col-lection and refinement statistics are summarized in Table S1. Figures wereprepared with PyMOL (www.pymol.org).

Binding Assays. Protein interactions were tested using both SPR analysisand the Octet RED96 biosensor method. For the SPR assays, a BIAcore3000 spectrometer was used to measure kinetic constants at room temper-ature (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 wereimmobilized on CM5 chips (GE Healthcare) at approximately 1,000 responseunits and analyzed for real-time binding by flowing through gradient con-centrations (ranging from 7.8 to 1,000 nM) of Gn head proteins. For theOctet RED96 biosensor method, samples of buffer were dispensed intopolypropylene 96-well black flat-bottom plates (Greiner Bio-One) at a vol-ume of 200 μL per well, and all measurements were performed at 298 K inHBS-EP buffer with the plate shaking at the speed of 1,000 rpm. Anti-humanIgG Fc (AMC)-coated biosensor tips (Pall ForteBio) were used to captureantibody 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 measurementsfor antigen binding were performed by exposing biosensors to a series ofanalyte concentrations (15.6−250 nM for Gn wild-type, and 15.6–500 nM forGn mutants) and background subtraction was used to correct for sensordrifting. All sensors were generated with 10 mM Glycine-HCl, (pH 1.7, GEHealthcare). The data were processed by ForteBio’s data analysis softwareand plotted by Origin software.

Neutralization Assay. Fifty-microliters of twofold serial-diluted MAb 4–5 orcontrol antibody (Ebola monoclonal antibody 13C6) (57) was mixed with equalvolume of 100 TCID50 SFTSV (SDYY007), or RVFV (BJ01) at 310 K for 1 h. Thevirus–antibody mixture was then transferred to 80% confluent Vero cells in a96-well plate and incubated at 310 K for 1 h. The plate was washed with DMEMthree 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 havingneutralizing capacity if 100% of viral cytopathic effects was inhibited.

Identification of Protein Disulfide Bonds by MS Analysis. Purified SFTSV Gn-Cwas denatured in a buffer containing 8 M Urea, 1 mM Tris, pH 6.5, 2 mMN-ethylmaleimide before it was subjected to SDS/PAGE. The monomer anddimer bands of Gn-C were excised and digested in gel according to a pre-viously published protocol (58), with the following modifications: (i) re-duction and alkylation were omitted, (ii) 0.5 mM N-ethylmaleimide wasadded to all of the destaining solutions and wash buffers, and (iii) dehy-drated gel slices were rehydrated with 100 mM Tris, pH 6.5, containing Lys-Cand Asp-N at 5 ng/μL each for overnight digestion. Digested peptides wereanalyzed on a Q Exactive mass spectrometer coupled to an Easy Nano-LC1000 liquid chromatography system (Thermo Fisher Scientific).

Peptides were desalted on a 75-μm × 6-cm precolumn that was packed with10 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 (WelchMaterials). 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, thenmaintaining at 80% buffer B for 7 min. The flow rate was 200 nL/min. The MSparameters were as follows: the top 20 most-intense ions were selected for HCDdissociation; R = 140,000 in full scan, R = 17,500 in HCD scan; AGC targets were1e6 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 searchedusing pLink (60) against a protein database containing the sequences of theanalyte protein and all of the proteases used. The pLink parameters searchwere maximum number of missed cleavages = 5; minimum peptide length =4 amino acids; fixed modification of −1.007285 Da on cysteine and the disulfidemass was set to zero to allow the identification of more than one disulfidebond in a peptide pair; candidate pairs satisfying Mα+Mβ+Mlinker <M ± 5 Dawere scored for spectrum-peptide matching (Mα, Mβ, Mlinker, and M denotethe masses of the candidate α-peptide, candidate β-peptide, the linker, and theobserved precursor, respectively). pLink search results were filtered by requiringE-value < 0.001, false-discovery rate < 0.05, spectra count ≥ 20, and no morethan 10-ppm deviation between the monoisotopic masses of the observedprecursor and the matched disulfide-linked peptide (or peptide pair).

Western Blot Analysis. The SFTSV and RVFV Gn head proteins were fractionatedby 12% SDS/PAGE. The separate proteins were electro-transferred to a nitro-cellulose membrane and incubated with full-length MAb 4–5 antibody and ahorseradish peroxidase-conjugated secondary goat anti-human IgG antibody (SC-2453; Santa Cruz). SuperSignal West Pico Chemiluminescent Substrate (Pierce34080) was used for detection according to the manufacturer’s instructions.

Fitting of the RVFV Gn Structure into the Low-Resolution EM Structure. Thecrystal structure of RVFV Gn was manually fitted into the capsomers in the22-Å resolution cyro-EM map of RVFV (EMDataBank ID code EMD-1550) withUCSF Chimera (61). The positions of Gn with the five- and six-coordinatedcapsomers were adjusted according to the fivefold and sixfold rotationalsymmetry, respectively, without major steric clashes.

ACKNOWLEDGMENTS. We thank Dr. Zheng Fan for technical help withBiacore experiments. This work was supported by a China National GrandS&T Special Project (No. 2017ZX10303403); the Strategic Priority Research Pro-gram of the Chinese Academy of Sciences (Grants XDPB03 and XDB08020100);and the National Natural Science Foundation of China (Grants 81330082,81301465, and 31570926). Y.W. is supported by Chinese Academy of SciencesYouth Innovation Promotion Association Grant 2016086. G.F.G. is a leadingprincipal investigator of the National Natural Science Foundation of ChinaInnovative Research Group (Grant 81621091).

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