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

Yan 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,NaLii, 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, Beijing 100101, China; bShenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, Shenzhen 518112, China; cState Key Laboratory of Agrobiotechnology, 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 Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Key Laboratory of Enteric Pathogenic Microbiology, Ministry Health, Nanjing 210009, China; gCenter for Influenza Research and Early-Warning, Chinese Academy of Sciences, Beijing 100101, China; hNational Institute of Biological Sciences, Beijing 102206, China; iNational Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China; jNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; and kResearch Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of 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) and RVFV was isolated in Kenya in 1930 (9, 10). Recurring out- Rift Valley fever virus (RVFV) are two arthropod-borne phlebovi- breaks of RVFV disease have been reported in ruminants and ruses in the Bunyaviridae family, which cause severe illness in humans in Africa and the Arabian Peninsula (11, 12), consti- humans and animals. Glycoprotein N (Gn) is one of the envelope tuting a significant threat to global public health and agriculture. proteins on the virus surface and is a major antigenic component. Humans can be infected by bites from virus-carrying mosquitoes Despite its importance for virus entry and fusion, the molecular or through contact with bodily fluids of the infected animals (13). features of the phleboviruse Gn were unknown. Here, we present

RVF patients display a self-limiting febrile illness, and some MICROBIOLOGY the crystal structures of the Gn head domain from both SFTSV and cases may develop lethal hemorrhagic fever, neurologic disor- RVFV, which display a similar compact triangular shape overall, ders, or blindness (13). Vaccines have been used to control RVF while the three subdomains (domains I, II, and III) making up the among livestock in endemic regions in Africa and the Arabian Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are Peninsula (14, 15). However, formalin-inactivated whole-virus vac- responsible for Gn dimerization, as revealed in this study, and they cines show little immunogenicity (16, 17), whereas live-attenuated are highly conserved for all members in Bunyaviridae. Therefore, vaccine is teratogenic in pregnant sheep and cattle (18, 19). Cur- we propose an anchoring mode on the viral surface. The complex rently, there are no effective vaccines or antiviral agents approved structure of the SFTSV Gn head and human neutralizing antibody for use in humans. MAb 4–5 reveals that helices α6 in subdomain III is the key com- ponent for neutralization. Importantly, the structure indicates that Significance domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neu- Bunyaviruses are emerging zoonotic pathogens of public- tralizing antibodies. Collectively, Gn is a desirable vaccine target, health concern. Lack of structures for proteins on the viral and our data provide a molecular basis for the rational design of membrane (“envelope”) surface limits understanding of entry. vaccines against the diseases caused by phleboviruses and a model We describe atomic-level structures for the globular “head” of for bunyavirus Gn embedding on the viral surface. the envelope protein, glycoprotein N (Gn), from two members, severe fever with thrombocytopenia syndrome virus (SFTSV) bunyavirus | SFTSV | glycoprotein | neutralizing antibody | RVFV and Rift Valley fever virus (RVFV), of Phleboviruses genus in the bunyavirus family, and a structure of the SFTSV Gn bound with he Bunyaviridae is a large family of human, animal, and plant a neutralizing antibody Fab. The results show the folded Gn Tpathogens spanning five genera: Orthobunyavirus, Hantavirus, structure and define virus-specific neutralizing-antibody bind- Nairovirus, Phlebovirus,andTospovirus (1). With the exception of ing sites. Biochemical assays suggest that dimerization, medi- hantaviruses, all other bunyaviruses are arthropod-borne viruses. ated by conserved cysteines in the region (“stem”) connecting Severe fever with thrombocytopenia syndrome virus (SFTSV) and the Gn head with the transmembrane domain, is a general Rift Valley fever virus (RVFV) belong to the Phlebovirus genus, feature of bunyavirus envelope proteins and that the dimer is which can cause emerging infectious diseases in humans (1–3), as probably the olimeric form on the viral surface. emphasized by the recent imported case of RVFV infection in China after its first emergence in Africa over 80 y ago (4). Author contributions: Y.W. and G.F.G. designed research; Y.W., Y. Zhu, F.G., B.O.O., Y.B., SFTS cases were first reported in China during 2007; however, 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., and the causative agent was not isolated from patients who presented G.F.G. analyzed data; and Y.W., F.G., G.W., and G.F.G. wrote the paper. with fever, thrombocytopenia, leukocytopenia, and multiorgan The authors declare no conflict of interest. dysfunction until 2011 (2, 5). SFTSV-infected patients have been This article is a PNAS Direct Submission. found in at least 13 provinces in China, with a case fatality rate of ≈ Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, 12%. In 2013, cases of SFTS were reported in South Korea and www.wwpdb.org (PDB ID codes: 5Y0W, 5Y0Y, 5Y10, and 5Y11). Japan, with a case fatality rate of 35.4% and 50%, respectively 1Y.W., Y. Zhu, F.G., and Y.J. contributed equally to this work. – (6 8). Unfortunately, vaccine or antiviral intervention against 2To whom correspondence may be addressed. Email: [email protected] or gaof@ SFTSV remain unavailable. im.ac.cn. RVFV is an emerging mosquito-borne zoonotic infectious This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pathogen and the prototype virus of the Phlebovirus genus (1). 1073/pnas.1705176114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1705176114 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 As with the other members of the Bunyaviridae family, SFTSV of the SFTSV Gn head domain, and succeeded in obtaining its and RVFV genomes contain three negative-stranded RNA seg- crystal structure. Both the crystal structures of SFTSV and RVFV ments (L, M, and S) (1). The M segment encodes a glycoprotein Gn head domains were determined at a resolution of 2.6 Å (Table precursor, which can be cleaved by cellular proteases during S1). A Dali search within the Protein Data Bank (PDB) failed to translation (20). For SFTSV, the precursor can be processed identify any existing structures to the Gn of both SFTSV and into two subunits: glycoprotein N (Gn) and glycoprotein C (Gc) RVFV, suggesting a novel fold. The structure of SFTSV Gn was (1), while for RVFV, one more subunit, called nonstructural solved by “antibody-walking” (Materials and Methods), in which the protein in the M segment is processed, in addition to Gn and Gc. antibody provides model-based phasing information to determine Gn/Gc are responsible for attachment and membrane fusion, the unknown structure. The RVFV Gn structure was solved by which is required for host cell entry (21–23). The recently solved molecular replacement, using SFTSV Gn as a search model. crystal structures of RVFV and SFTSV Gc proteins revealed The Gn head domains of both SFTSV and RVFV fold into a architectural similarity with class II viral fusion proteins (24, 25). similar triangular shape architecture consisting of three sub- Low pH can induce RVFV Gc oligomerization, but has no in- domains (Fig. 1 B and C). Subdomains I and II form the foun- fluence on Gn (23). Recent studies reveal that the lectin dendritic- dation bed supporting the subdomain III protruding on the top cell (DC) specific intercellular adhesion molecule 3-grabbing (Fig. 1 B and C and Fig. S1A). However, the three subdomains nonintegrin (SIGN) is identified as the entry factor required for show a different arrangement between these two viruses, with an many phleboviruses, including SFTSV, RVFV, Toscana virus (TOSV), average rmsd of 5.010, 2.393, and 1.825 for subdomains I, II, and and Uukuniemi virus (UU.K.V) (26, 27). L-SIGN, another C-type III, respectively (Fig. S1B). Specifically, in subdomain I of lectin, shares 77% sequence homology with DC-SIGN and acts as SFTSV, a five-stranded β-sheet (β2, β3, β4, β5, and β9), one helix an attachment receptor for these phleboviruses, rather than as an (α3), and one 310-helix (η2) are located on the interface of endocytic receptor (28). Nonmuscle myosin heavy chain II A subdomains I and II. Two α-helices (α1 and α2), three 310-helices (NMMHC-IIA) is reported as a critical factor contributing to the (η1, η3, and η4), and two sets of small antiparallel β-strands efficiency of early SFTSV infection, and the recombinant Gn (β1 and β8, β6 and β7) flank the β-sheet on the opposite side. A protein is capable of binding NMMHC-IIA, indicating that Gn is free cysteine (C99) on α3 is located in the interior of subdomain likely the key receptor-binding protein (26). I. For the RVFV Gn subdomain I, a four-stranded β-sheet (β1, Gn and Gc are two major antigenic components on the viral β2, β3, and β6) and three α-helices (α2, α3, and α4) can be found surface and are the targets of specific neutralizing antibodies on the interface of subdomains I and II. One α-helix (α1), three (29). MAb 4–5 is a human-origin, neutralizing monoclonal an- 310-helices (η1, η2, and η3), and one pair of small antiparallel tibody targeting Gn, showing cross-neutralizing activity to a wide β-strand (β4 and β5) are on the opposite side. β-Strands are the range of SFTSV isolates in China (30). An RVFV Gn/Gc subunit major component in subdomain II and the pattern of β-strands vaccine was previously shown to elicit a strong neutralizing an- between SFTSV and RVFV is the same (Fig. 1 D and E and Fig. tibody response in sheep (31). S2). The core structure comprising the six β-strands is located Here, we report the crystal structures of two Gn head domains next to subdomain I and a three-stranded β-sheet connects to (from SFTSV and RVFV) and the SFTSV Gn head domain in subdomain III. The only different secondary element in sub- complex with MAb 4–5, a neutralizing antibody identified in SFTS domain II between these two is the α-helix. SFTSV contains an recovered patients (30). These two Gn head domains display a α4, whereas RVFV does not have it in subdomain II. The sec- similar overall configuration but a different overall topology to the ondary elements in subdomain III between SFTSV and RVFV Gn head structure of Puumala hantavirus (PUUV) (32). Four are similar. Four β-strands and three α-helices stabilize sub- cysteine residues highly conserved in the stem region of Gn are domain III, in which α5 in SFTSV is replaced by η5 in RVFV responsible for dimerization, suggesting the Gn cysteine-mediated (Fig. 1 D and E). Although the Gn structures in the Phlebovirus dimer model might apply to the entire Bunyaviridae family. The genus display similar configurations, the overall structures be- complex structure reveals that the key residues in SFTSV recog- tween the Phlebovirus genus and Hantavirus genus are distinct nized by neutralizing MAb 4–5 is not conserved in RVFV; there- (Fig. S1 C and D). Furthermore, the topology between these two fore, MAb 4–5 cannot bind to RVFV Gn. Moreover, conserved genera is completely different (Fig. S1E). exposure amino acid alignment of 11 members in Phlebovirus genus The glycosylation sites are observed to be different between indicates that domain III of Gn provide epitopes for specific SFTSV and RVFV Gn. For SFTSV, two N-linked glycans neutralizing antibody, while domain II is probably an ideal region (N33 and N63) can be observed in subdomain I (Fig. 1 A, B, and recognized by a broadly neutralizing antibody. Altogether our D and Fig. S2), which is consistent with theoretical predictions. findings have proved that Gn is a promising antigen for vaccine For RVFV, although N438 is predicted to be an N-linked gly- development and the crystal structures provide a molecular basis cosylation site, no glycans can be observed in the solved crystal for the rational design of vaccines and antiviral drugs. structure in the insect cell-expressed protein (Fig. 1 A, C, and E). Both the SFTSV and RVFV Gn are cysteine-rich proteins. Results SFTSV Gn has 27 cysteines, whereas RVFV Gn has 28 cysteines. Overall Structure of the Gn Head Domain: SFTSV and RVFV. Both Twelve cysteines in the head domains are identical between these SFTSV and RVFV Gn are type I transmembrane proteins with two Gn, and all of the other 10 cysteines in the stem domains are the N-terminal ectodomain binding on the cell surface and a conserved for the 11 sequences of the available phleboviruses (Figs. C-terminal transmembrane helix anchored on the virus mem- S2 and S3A). The SFTSV Gn head domain contains eight disulfide brane (Fig. 1A). The ectodomain can be divided into the head bonds and a free cysteine. Specifically, two disulfide bonds (C26- and stem domains. To facilitate crystallization, the stem region C49 and C143-C156) and an unpaired cysteine (C99) are in sub- of SFTSV Gn was removed by limited trypsin digestion due to an domain I, one disulfide bond is in subdomain II (C206-C216), one unsuccessful effort with the full-length ectodomain for crystalli- disulfide bond is across subdomains II and III (C180-C327), and zation. The last amino acid visible in the crystal structure is four disulfide bonds are in subdomain III (C258-C305, C266-303, N340. However, the molecular weight measured by mass spec- C274-C280, and C287-C292). The RVFV Gn head domain is sta- trometry (MS) indicates that the cleavage site is between resi- bilized by nine disulfide bonds, four of which are in subdomain I dues K371 and S372, suggesting a conformational disorder in the (C179-C188, C229-C239, C250-C281, and C271-C284), one of C terminus of the truncated Gn (residues 340–372), instead of which is in subdomain II (C322-C332), three of which are in sub- degradation during crystallization (Fig. 1A). We designed the domain III (C374-C434, C402-C413, and C420-C425), and one of construct of the RVFV Gn head domain based on the structure which is across subdomains II and III (C304-C456). Six disulfide

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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 fol- lowing 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).

bonds (C271-C284 in subdomain I, C304-C456 in subdomain II, in solution, respectively (Fig. 2A). Nonreducing SDS/PAGE C322-C332 crossing subdomain II and III, C374-C434, C402-C413, shows that the dimer is linked by disulfide bonds (Fig. S4A). and C420-C425 in subdomain III, RVFV Gn numbering) are con- Notably, the dimer was completely disassociated after a treat- served between the SFTSV and RVFV Gn head domains (Fig. S2). ment of limited trypsin proteolysis (Fig. 2A and Fig. S4A), indicating that the C terminus of Gn is solely responsible for Dimerization of Gn Through Its Stem Region. The full-length SFTSV dimerization via disulfide bonds (Fig. 2A). To characterize the Gn ectodomain was produced using the baculovirus expression C terminus of Gn, we constructed the unstructured part of Gn system. Results from size-exclusion chromatography using a (Gn-C), which corresponds to residues 338–452. The Gn-C protein Superdex 200 10/300 GL column showed the peaks eluted at forms a dimer under nonreducing conditions, which is consis- 12.6 mL and 14.4 mL, corresponding to the dimer and monomer tent with the above-described result (Fig. S4B). Full-length

Wu et al. PNAS Early Edition | 3of10 Downloaded by guest on October 1, 2021 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.

RVFV Gn forms a dimer under nonreducing conditions as well hemagglutinin antibody CR8020 (IgG1) (33). To verify neutralizing (Fig. S4C). activity, we also constructed full-length MAb 4–5 with human Ig To determine which cysteines are responsible for dimerization, G1 (IgG1). The recombinant antibodies were expressed by 293T we used MS and site-directed mutagenesis to analyze Gn-C and mammaliancellsandtheconcentrationrequiredtoobtain50% the full-length Gn protein of SFTSV. MS analysis identified four neutralization of 100 TCID50 SFTSV in vitro was 44.2 μg/mL (Fig. intermolecular disulfide bonds involving C430, C435, C438, and 3A). The interaction between Gn and MAb 4–5wasfurther C447 from the Gn-C dimer but not the monomer (Table S2), demonstrated by surface plasmon resonance (SPR) assays with a suggesting that these cysteine residues may mediate Gn di- dissociation constant (Kd) of 25.9 nM (Fig. 3B). We also measured merization. Indeed, simultaneously mutating these four cysteine the binding between RVFV Gn and MAb 4–5, but no binding or residues to alanine completely abolished dimer formation of the neutralization was observed, indicating the specific binding of MAb full-length Gn, leaving only the monomer (Fig. 2B). Further- 4–5toSFTSV(Fig.3C and D). more, the C430A/C447A substitution shifted the equilibrium toward the Gn monomer, but failed to abolish the Gn dimer. The Complex Structure of the SFTSV Gn Head and Neutralizing MAb 4–5. C435A/C438A substitution reduced the relative abundance of To elucidate the structural basis of virus neutralization, we fur- the dimer more substantially than the C430A/C447A substitu- ther prepared the Gn head–MAb 4–5 Fab complex by mixing the tion (Fig. 2B), suggesting that although C430, C435, C438, and two proteins in vitro and then purifying by size-exclusion chro- C447 all contribute to Gn dimerization, the two intermolecular matography (Fig. S8). Consistent with the high binding affinity disulfide bonds mediated by C435 and C438 play a major role. between Gn head and MAb 4–5, the complex is stable and easily MS analysis also identified a disulfide bond between C356 and obtained. Crystals diffracting to 2.1 Å were grown from digested C424 in both the Gn-C monomer and dimer (Table S2). Muta- Gn in complex with MAb 4–5 Fab (Table S1). The complex tion of either C356 or C424 severely reduced the yield of the structure was determined by molecular replacement using a Fab protein (Fig. S5), suggesting that the C356-C424 disulfide bond is structure (PDB ID code 4RIR) as the search model followed by critical for stabilizing Gn. The data described above support that iterative rounds of model building and refinement. Automatic the cysteines in the stem region are responsible for dimerization model extension of the missing domain of the Gn head was and the cysteines are highly conserved in the same genus (Figs. carried out with AUTOBUILD in PHENIX (34). Therefore, the S3 and S6). It is noteworthy that the last four cysteines (C430, SFTSV Gn structure described earlier was actually solved by C435, C438, and C447, SFTSV numbering) responsible for di- “antibody-walking.” Structure analysis reveals that MAb 4–5 merization are highly conserved across five genera (Phlebovirus, binds the membrane-distal head of Gn and the contact was Hantavirus, Nairovirus, Orthobunyavirus, and Tospovirus) of the mediated only by the heavy chain (Fig. 4A). The interaction Bunyaviridae family, indicating that members in the whole surface between MAb 4–5 and Gn buries 614.8 Å2 of the mo- Bunyaviridae likely share the same assembly organization with a lecular surface. The primary region of the interaction is the Gn disulfide bond-linked dimer (Fig. S7). complementarity-determining region (CDR) H3 (Fig. 4B), which penetrates the hydrophobic trough, consisting of α5, α6, and Human Antibody MAb4-5 Specificity to SFTSV Gn. Antibody MAb 4–5 η5 in SFTSV Gn domain III. In this interaction, Y104 of the was previously isolated using whole SFTSV virions as bait from a CDR H3 interacts with F256, F286, V289, and A290. Notably, phage-display antibody library derived from the peripheral blood α6 is the key element on the Gn head domain, which contacts all mononuclear cells of a patient that recovered from SFTS disease three CDR regions in the MAb 4–5 heavy chain. In particular, (30). Since only the sequence of the variable region was available, K288 is the most important residue located on α6. Specifically, we constructed the Fab fragment with the variable region of MAb the K288 main chain hydrogen-bonds the W33 side chain on the 4–5 heavy and light chains, combined with the constant region of a CDR H1 loop. The side chain of K288 can form salt bridges with

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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.

the side chains of both D55 and D57 on the CDR H2 loop. replaced by Y423 in RVFV. Additionally, the side chain of K288 main chain hydrogen bonds the side chain of R101 on the K405 displays steric hindrance to CDR H3 loop. On the other CDR H3 loop. Moreover, two electrostatic patches are observed hand, K405 in RVFV is a positive-charged amino acid, which on the antigen–antibody interface. The basic patches, which consist shows repulsive force to the positive-charged R102 on the CDR of K100 and R102 on the CDR H3 loop, interact with E293 on Gn, H3 loop. Further conservation analysis of surface amino acids while the acidic patch (D55 and D57) on the CDR H2 loop forms among 11 phleboviruses indicates that domain III is a variable salt bridges with Gn K288 (Fig. 4C). To determine the critical role region, which can be recognized by a specific neutralizing anti- of residue 288 in MAb 4–5 binding, two substitutions (K288A and body (Fig. S10A). In contrast, domain II shows the relatively K288E) were constructed, expressed, and purified. Affinity assay conserved epitope, which may be recognized by broadly neu- indicates that these two substitutions dramatically decrease the tralizing antibody (Fig. S10 B and C). binding between Gn and MAb 4–5(Fig. S9). The MAb 4–5 can recognize the denatured SFTSV Gn head Discussion but not the RVFV Gn head by Western blot (Fig. 5A). Structural In this study, we have reported two Gn head structures, each with analysis indicates that α6 is the major epitope recognized by a distinct architecture. Interestingly, a recent Gn structure from MAb 4–5. Comparison of the epitope amino acids sequence PUUV (32) is topologically different from the structures reported between SFTSV and RVFV shows that only two amino acids are here. This may imply that viruses from different genera in the conserved (F286 and C287, SFTSV numbering) (Fig. 5B). The Bunyaviridae family may have different virus envelope protein key residue K288 is replaced by serine in RVFV, which is the architecture, which would be an interesting topic to be studied in main reason leading the abolishment of binding to MAb 4–5. the future. Specifically, the salt bridges between K288 and D55, K288 and So far, the real pattern of the arrangement of viral glycoproteins D57 were lost. The hydrogen bonds between the Gn head and on the virus surface (how Gn and Gc, and other members if any, heavy chain were lost due to the shift of the main chain (Fig. 5C). assemble into a functional complex) has not been determined Moreover, the hydrophobic pocket in SFTSV Gn can accom- clearly for any members of the bunyaviruses, even though a study modate the side chain of F104 in the MAb 4–5 heavy chain. In proposing a low-resolution model was recently published (32). contrast, the pocket becomes shallow (Fig. 5D), as A290 is Previous studies have shown the Gn and Gc in several members of

Wu et al. PNAS Early Edition | 5of10 Downloaded by guest on October 1, 2021 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.

the phleboviruses—such as RVFV (35), UU.K.V (36), and Punta possibility that dimerization of viral surface proteins plays an im- Toro virus (PTV) (37)—can be isolated as a heterodimer from portant role in attachment and entry into the host cell. Based on the mature virions or the infected cells. The Gn/Gc heterodimer may biochemical assays, we propose here that the Gn dimer linked by the further assemble to form higher-ordered assemblies or “spikes” on C-terminus disulfide bonds is likely the basic unit on the virus sur- the virion surface for proper transit to the Golgi apparatus (38), face, and further assembles to form higher-ordered organization and a tetrameric model was proposed in the recent study of PUUV (Fig. 6A). To prove our model, the crystal structure of RVFV Gn (32). However, Gn homodimers have also been isolated from UU.K. wasfittedintotheT= 12 icosahedral cryo-EM map of RVFV (44) V virions (39), indicating that this kind of assembly cannot be ruled (Fig. 6B). There are five Gn molecules in each of 12 five-coordinated out. Moreover, disulfide-linked viral glycoprotein dimers were also capsomers and six Gn molecules in each of 110 six-coordinated reported in other related viruses: for example, measles virus hem- capsomers in the glycoprotein shell of RVFV (Fig. 6 B and C). agglutinin in measles virus (40–42) and hemagglutinin-esterase Each pair of closest Gn molecules from two neighboring capsomers protein in the subset of the betacoronaviruses (43). This raises the form a Gn dimer (Fig. 6 C and D). There are 360 Gn dimers in one

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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.

RVFV particle in total. Moreover, the cryo-EM structure-fitting data HRTV is arginine, which is also a positively charged amino acid, of both RVFV and PUUV show that Gc occupies the inner half of suggesting HRTV Gn may be capable of binding to MAb 4–5, the glycoprotein shell, while Gn is exposed outside, indicating that and being neutralized. For other phleboviruses, the corre- Gn is the major target for antibodies after infection. Additionally, it is sponding residues are negative-charged amino acids (glutamine) clear that the structure of all solved Gc display typical characteristics or uncharged hydrophilic amino acids (serine, threonine, and of a class II fusion protein (24, 25, 45, 46), and the Gc have played a asparagine), which may not be neutralized by MAb 4–5. More- key role in fusion process. Gn likely contributes to receptor binding over, E293 (SFTSV numbering) is another important residue in according to its position on the viral surface and our work reported MAb 4–5 binding, which has electrostatic interaction with a here. However, determining the real function of the SFTSV Gn positive-charged group (K100, R101, and R102) in the CDR protein and the organization of two envelope proteins (Gn and Gc) H3 loop. However, not all of the residues in other phleboviruses within the virions will require high-resolution cryo-EM data and the are negative-charged amino acids. Take RVFV as an example: the structural characterization of Gn/Gc complex. corresponding residue is lysine (K), which has electrostatic re- Another interesting question is whether the MAb 4–5 has pulsion to the positive-charged group in CDR H3 loop. broad neutralizing capacity for the members of the Phlebovirus Although no specific host cell receptors have been identified in genus in Bunyaviridae family. Members in the Phlebovirus genus this genus thus far, the complex structure of Gn with its neutralizing can initially be classified into two groups: the Sandfly fever group antibody MAb 4–5 implies that the receptor binding site is likely and the Uukuniemi group based on the antigenic, genomic, and located around the α6 helix in SFTSV. Alignment of the α6 helix in vector relationships (47). RVFV belongs to the Sandfly fever phleboviruses indicates that only C287 is conserved. Moreover, cell group. Phylogenetic studies indicate that SFTSV represents a third tropism among these viruses are not identical as well, suggesting distinct group within the Phlebovirus genus, which is transmitted by their differences in receptor requirements. Specifically, RVFV can ticks (2). Furthermore, another phlebovirus, Heartland virus (HRTV), infect the thymus, spleen, and liver. FACS analysis in RVFV-GFP– which was isolated from humans during 2012 in the United States, is infected mice showed that the macrophages, DCs, and granuclocytes closely related to SFTSV (48), with 61.9% amino acid identity in the were the main target cells for RVFV (49). SFTSV Gn/Gc pseudogtypes M segment. K288 (SFTSV numbering) of SFTSV Gn is the key res- infected human lung (BEAS-2B, A549 and H1299), kidney (293T), idue involved in MAb 4–5 binding. The corresponding residue in liver (HepG2), colon (Caco-2), retinal epithelium (RPE), and

Wu et al. PNAS Early Edition | 7of10 Downloaded by guest on October 1, 2021 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.

glioblastroma (U373) cell lines, as well as human monocyte-derived recombinant proteins were produced in High Five cells. The cell culture media DCs. Monocytic (THP-1) cells HFF and cervical carcinoma (HeLa) were collected 60 h after infection and then purified by nickel affinity chro- cells were resistant to SFTSV (27). matography 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, Our work represents structures of Gn proteins in the Phlebovirus Bunyaviridae pH 8.0, 50 mM NaCl. Gn protein was pooled and incubated with trypsin at genus of the family, and provides a detailed view of a mass ratio of 300:1 at 277 K overnight, and further purified on a Superdex − the interaction between SFTSV Gn and a neutralizing antibody. 200 column (GE Healthcare). Digested Gn was then concentrated to 7.5 mg mL 1 The Gn in RVFV and SFTSV display a novel fold, and four cys- for crystallization. The RVFV Gn head domain (GenBank accession no. teines in the C terminus play important roles in dimerization among JQ068143.1, residues 154–469) was constructed using the same strategy with- −1 members in the whole Bunyaviridae. Moreover, the complex struc- out trypsin digestion. The Gn head was concentrated to 10 mg mL for crys- ture provides important information for the immune epitope, which tallization. To prove the C terminus of Gn playing an important role in dimerization, the Gn-C (residues 338–452) was constructed using the same may have implications for design of vaccines that are capable of strategy as the SFTSV Gn ectodomain described above. Moreover, the expres- eliciting effective immune responses against phleboviruses. 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 constant Materials and Methods region of CR8020 IgG1 (33) into the pcDNA4 expression vector. A six-histidine Protein Expression and Purification. The SFTSV Gn ectodomain (GenBank ac- tag was designed at the C terminus of the light chain for purification. The MAb cession no. JF906057.1, residues 20–452) followed by a C-terminal six-histidine 4–5 heavy chain was subcloned into a modified pcDNA4 with mouse IgG Fc purification tag was subcloned into the pFastBac1 vector (Invitrogen) modified at the C terminus. The full-length and the Fab fragment of MAb 4–5were with a gp67 signal sequence at the N terminus, as previously described (50–53). produced by HEK293T cells and purified by protein A and HisTrap HP column, Transfection and virus amplification were conducted with sf9 cells, and the respectively. MAb 4–5 Fab was further purified on a Superdex 200 column in

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1705176114 Wu et al. Downloaded by guest on October 1, 2021 20 mM Tris, pH 8.0, 50 mM NaCl for crystallization, while the full-length MAb 4–5 were observed every 24 h for 6 d. The antibody was considered as having PNAS PLUS was purified on a Superdex 200 column in PBS buffer for neutralization assay. neutralizing capacity if 100% of viral cytopathic effects was inhibited. 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 Identification of Protein Disulfide Bonds by MS Analysis. Purified SFTSV Gn-C mixture was then loaded on a Superdex 200 column in 20 mM Tris, pH 8.0, was denatured in a buffer containing 8 M Urea, 1 mM Tris, pH 6.5, 2 mM −1 50 mM NaCl, and concentrated to 10 mg mL for crystallization. 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 pre- Crystallization. All crystallizations were performed using a vapor-diffusion viously published protocol (58), with the following modifications: (i)re- sitting-drop method with 1 μL protein mixing with 1 μL reservoir solution. duction and alkylation were omitted, (ii)0.5mMN-ethylmaleimide was SFTSV Gn crystals were grown in the reservoir solution comprising of 20% added to all of the destaining solutions and wash buffers, and (iii) dehy- (wt/vol) PEG 4000, 20% (vol/vol) 2-propanol, and 0.1 M sodium citrate, pH 5.5, drated gel slices were rehydrated with 100 mM Tris, pH 6.5, containing Lys-C at 291 K. RVFV Gn head crystals were grown in the reservoir solution of 0.2 M and Asp-N at 5 ng/μL each for overnight digestion. Digested peptides were ammonium sulfate, 0.1 M Mes, pH 6.5, 20% (wt/vol) PEG 8000 at 277 K. Good analyzed on a Q Exactive mass spectrometer coupled to an Easy Nano-LC diffraction crystals of the complex protein were finally obtained in 0.3 M po- 1000 liquid chromatography system (Thermo Fisher Scientific). tassium/sodium tartrate, 20% (wt/vol) PEG 3350, and 0.1 M Bis Tris propane, Peptides were desalted on a 75-μm × 6-cm precolumn that was packed with −1 pH 7.5, with protein concentration of 15 mg mL . Crystals were frozen in liquid 10 μm, 120 Å ODS-AQ C18 resin (YMC Co., Ltd.) and connected to a 75-μm × nitrogen in reservoir solution supplemented with 20% glycerol (vol/vol) as a 10-cm analytical column packed with 1.8 μm, 120 Å UHPLC-XB-C18 resin (Welch cryoprotectant. Data were collected at the Shanghai Synchrotron Radiation Materials). The peptides were separated over a 34-min linear gradient from 5% Facility BL17U. All data were processed with HKL2000. 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 Structure Determination and Refinement. The structure of the SFTSV Gn head- maintaining at 80% buffer B for 7 min. The flow rate was 200 nL/min. The MS – MAb 4 5 complex was determined by molecular replacement using an an- parameters were as follows: the top 20 most-intense ions were selected for HCD tibody Fab fragment (PDB ID code 4RIR) as search probe. The molecular dissociation; R = 140,000 in full scan, R = 17,500 in HCD scan; AGC targets were model was rebuilt using COOT (54) and refined with REFMAC (55). Sub- 1e6 for FTMS full scan, 5e4 for MS2; minimal signal threshold for MS2 = 4e4; sequently, the automatic model extension of the missing Gn domain was precursors having a charge state of +1, >+8, or unassigned were excluded; carried out with AUTOBUILD in PHENIX (34). The method used for de- normalized collision energy, 30; peptide match, preferred. termination of the unknown structure by getting the phasing information The raw data of the 10-protein sample were preprocessed using pParse (59), from antibody is called “antibody walking.” The SFTSV Gn head structure which was set to exclude coeluting precursor ions and precursors of +1, was solved by molecular replacement using the refined coordinates +2 charge. To identify disulfide-linked peptides, the MS2spectra was searched obtained from the complex and the structure was refined using REFMAC. using pLink (60) against a protein database containing the sequences of the The RVFV Gn head structure was solved by single-wavelength anomalous MICROBIOLOGY analyte protein and all of the proteases used. The pLink parameters search diffraction, with a gold derivative (NaAuCl ·2H O). Glycans were added at 4 2 were maximum number of missed cleavages = 5; minimum peptide length = the final stages of model building and refinement according to the density 4 amino acids; fixed modification of −1.007285 Da on cysteine and the disulfide map. Structure validation was performed with PROCHECK (56). Data col- mass was set to zero to allow the identification of more than one disulfide lection and refinement statistics are summarized in Table S1. Figures were bond in a peptide pair; candidate pairs satisfying Mα+ Mβ+ Mlinker < M ± 5Da prepared with PyMOL (www.pymol.org). 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 Binding Assays. Protein interactions were tested using both SPR analysis observed precursor, respectively). pLink search results were filtered by requiring and the Octet RED96 biosensor method. For the SPR assays, a BIAcore E-value < 0.001, false-discovery rate < 0.05, spectra count ≥ 20, and no more 3000 spectrometer was used to measure kinetic constants at room temper- than 10-ppm deviation between the monoisotopic masses of the observed ature (298 K). All proteins were exchanged into a buffer of 20 mM Hepes, precursor and the matched disulfide-linked peptide (or peptide pair). 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 Western Blot Analysis. The SFTSV and RVFV Gn head proteins were fractionated units and analyzed for real-time binding by flowing through gradient con- by 12% SDS/PAGE. The separate proteins were electro-transferred to a nitro- centrations (ranging from 7.8 to 1,000 nM) of Gn head proteins. For the cellulose membrane and incubated with full-length MAb 4–5 antibody and a Octet RED96 biosensor method, samples of buffer were dispensed into horseradish peroxidase-conjugated secondary goat anti-human IgG antibody (SC- polypropylene 96-well black flat-bottom plates (Greiner Bio-One) at a vol- 2453; Santa Cruz). SuperSignal West Pico Chemiluminescent Substrate (Pierce ume 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 34080) was used for detection according to the manufacturer s instructions. 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, Fitting of the RVFV Gn Structure into the Low-Resolution EM Structure. The pH 7.4, 150 mM NaCl and 0.005% (vol/vol) Tween 20. Kinetic measurements crystal structure of RVFV Gn was manually fitted into the capsomers in the for antigen binding were performed by exposing biosensors to a series of 22-Å resolution cyro-EM map of RVFV (EMDataBank ID code EMD-1550) with analyte concentrations (15.6−250 nM for Gn wild-type, and 15.6–500 nM for UCSF Chimera (61). The positions of Gn with the five- and six-coordinated Gn mutants) and background subtraction was used to correct for sensor capsomers were adjusted according to the fivefold and sixfold rotational drifting. All sensors were generated with 10 mM Glycine-HCl, (pH 1.7, GE symmetry, respectively, without major steric clashes. Healthcare). The data were processed by ForteBio’s data analysis software and plotted by Origin software. ACKNOWLEDGMENTS. We thank Dr. Zheng Fan for technical help with Biacore experiments. This work was supported by a China National Grand S&T Special Project (No. 2017ZX10303403); the Strategic Priority Research Pro- Neutralization Assay. Fifty-microliters of twofold serial-diluted MAb 4–5or gram of the Chinese Academy of Sciences (Grants XDPB03 and XDB08020100); control antibody (Ebola monoclonal antibody 13C6) (57) was mixed with equal and the National Natural Science Foundation of China (Grants 81330082, volume of 100 TCID50 SFTSV (SDYY007), or RVFV (BJ01) at 310 K for 1 h. The 81301465, and 31570926). Y.W. is supported by Chinese Academy of Sciences virus–antibody mixture was then transferred to 80% confluent Vero cells in a Youth Innovation Promotion Association Grant 2016086. G.F.G. is a leading 96-well plate and incubated at 310 K for 1 h. The plate was washed with DMEM principal investigator of the National Natural Science Foundation of China three times, and incubated at 310 K in a 5% CO2 incubator. Cytopathic effects Innovative Research Group (Grant 81621091).

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