CCHFV GP38 and GP85 interact with cell-surface glycosaminoglycans

CCHFV GP38 and GP85 interact with cell-surface glycosaminoglycans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CCHFV GP38 and GP85 interact with cell-surface glycosaminoglycans Olivier Reynard, Romain R. Vivès, Olga Makshakova, Estelle Gallice, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8680483/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Crimean Congo Hemorrhagic Fever Virus (CCHFV) is a negative-strand segmented RNA virus responsible for severe hemorrhagic fever in humans. The M genomic segment of CCHFV encodes a polyprotein precursor that is processed by cellular proteases into several structural and non-structural proteins. Among them, GP38 and its precursor GP85 are known to be secreted into the extracellular environment. We investigated their abilities to bind cells and we identified that they strongly bind to the cell plasma membrane through interaction with glycosaminoglycans. This interaction was mapped to a surface-exposed basic cluster that combines both a prototypical GAG-binding domain and linearly distant amino-acids. The present study describes for the first time the CCHFV GP38/85 proteins interaction with glycosaminoglycan and characterizes the interaction domain. Biological sciences/Biochemistry Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Structural biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Crimean Congo hemorrhagic fever virus (CCHFV) was discovered in 1944 and was identified as being the causative agent of severe hemorrhagic fever [18]. This virus belongs to the order Bunyaviridae , family Nairoviridae , genus Orthonairovirus . CCHFV is an arbovirus hosted by ticks, notably those from the genus Hyalomma . The geographical distribution of CCHFV overlaps the distribution area of those hard ticks that ranges from Africa, Asia, central Europe, eastern Mediterranean countries (Balkan countries and, Turkey), and more recently western Europe countries e.g. Spain and France [5, 17, 34]. The first symptoms arise 5–15 days after initial infection and consist of an acute febrile illness, which is followed by a severe phase generally including hemorrhagic symptoms and characterized by low platelet count, coagulopathy, acute liver infection, and the release of high amounts of pro-inflammatory mediators [5]. The disease leads to death in 5–40% of the cases. The genome of CCHFV is composed of three segments termed large (L), medium (M), and,small (S) which respectively encode for the RNA polymerase, the glycoproteins and the nucleoprotein (and NS). The glycoproteins are translated as a long polyprotein of nearly 1700 amino acids that is further processed by cellular proteases, notably furin and SKI-1 proteases [37, 46]. This proteolysis and the complex O- and N- glycosylations lead to the generation of 3 predicted non-structural glycoproteins: the mucin protein, GP38, and NSm, as well as two structural glycoproteins Gn and Gc involved in low-density lipoprotein receptor (LDLR) recognition and fusion [28, 37]. The mucin protein is a highly glycosylated disordered protein that contains a high amount of O-linked glycans and five N-glycans. Mucin and GP38 proteins (also named GP1) were characterized or predicted in most nairoviruses with different levels of O-glycosylations [23]. While the mucin protein sequence is of low amino-acid homology between CCHFV isolates, GP38, Gn, NsM and Gc are more conserved [23]. In the supernatant of cells transfected to express the M segment or infected with CCHFV, GP38 is mostly found in a free form, but also as a non-furin-digested precursor named GP85 containing both GP38 and the mucin protein [37]. In addition, western blot analysis of supernatants shows the presence of another, poorly characterized ~ 160 kDa protein, comprising both GP38 and mucin protein [37]. Although the functional role of GP38 during CCHFV infection remains unclear, monoclonal antibodies directed against GP38 protect non-human primates from CCHFV lethal challenge and a vaccine targeting against GP38 can protect against lethal infection, thus indicating that GP38 is a critical pathogenicity factor [15, 42]. Glycosaminoglycans are targeted by a wide range of pathogens including bacteria, parasites, and viruses [1, 9, 26, 43, 44]. This observation was initially documented in 1966 in a rabbit model of HSV1 infection in which heparin was shown to protect from infection [24]. Since then, GAGs are documented as a co-factor of infection for multiple viruses, including the more recent SARS-COV-2 [11]. Here we show that secreted GP38 and GP85 bind cell surface glycosaminoglycans (GAGs) and we identify the precise binding domain. Material and Methods Cells, reagents, and transfections. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium Glutamax (Thermo) supplemented with 10% fetal calf serum (FCS) and Normocin (Invivogen) at 37°C. Jurkat, THP1 and total lymphocytes were cultured in RPMI Glutamax (Thermo) 10% FCS. Cell transfections were performed using Jet Optimus (Polyplus) according to the manufacturer’s instructions at a ratio of 1:1 (plasmid:reagent). Plasmids and mutagenesis. The GP85 sequence was subcloned from plasmid pCDNA3 M Ibar10200 (Genbank AF467768.2) into vector phCMV with a 6xHis tag at the carboxy terminus. Aigai virus (AIGV, former CCHFV genogroup VI, AP92 strain [31]) M segment (Genbank DQ211625.1) was cloned by RT-PCR from RNA extracted from the supernatant of infected cells and GP85 was further subcloned into phCMV vector with a 6xHis tag at the carboxy terminus. Mutagenesis of GP38 plasmid was performed using the Q5 mutagenesis kit (New England Biolabs) according to the manufacturer’s instructions. Protein production and purification Culture medium from phCMV GP85-6xHIS transfected HEK 293Tcells was harvested 48 to 72 hours post-transfection, clarified by centrifugation (3000g, 5 minutes), and filtered through 0.22µm filter (Millipore). Tetradentate chelating agarose resin with divalent cobalt (Co2+) (Thermo) was used for the affinity purification step. Binding was performed in the presence of 5 mM imidazole. The resin was washed twice with a 30-bed volume of PBS 10mM Imidazole. Elution was performed by applying a 200 mM Imidazole solution in PBS pH 7.5. The proteins were further dialyzed against PBS in 5kDa cartridge (Thermo). The purified proteins contain an average of 85% of GP38, 12% of GP85 (precursor), and 3% of GP160. Flow cytometry analysis of GP38/85 binding Binding assay was perfomed on 10 5 cells per condition that were incubated on ice for 5 minutes, then spun for 1 minute at 750g and incubated with purified GP38/85 proteins (10µg/mL) for 30 minutes on ice. Cells were then washed twice with ice-cold PBS and stained using an anti 6xHIS antibody labeled with phycoerythrin (Miltenyi biotech) for 20 minutes on ice. Stained cells were washed twice with ice-cold PBS and analyzed directly on an LSR Fortessa 5L flow cytometer (Becton Dickinson). In silico simulations To identify the location of heparin-binding site, the molecular docking of heparin to GP38 was performed using ClusPro web server [29]. Then, complexes of GP38 and heparin were equilibrated using molecular dynamics simulations to take explicitly into account both ligand and protein flexibility in water solvent. The hexamer of heparan sulfate was placed in a proximity of the binding site, determined by molecular docking, and at the distance ~ 5Å from the protein surface to allow both protein and polysaccharide to adjust their geometries in the course of the complex formation. Molecular dynamics simulations were carried out in Amber 18 package[8], GLYCAM06j force field was used for heparin [40], generalized amber force field (gaff) for the chemical analog of GAG [48], and AMBER14SB for protein molecule [25]. The in silico site-directed mutagenesis was performed based on above described structures of native GP38 and HS complexes via a replacement of a target basic residue by glutamic acid. The new structures GP38 mutants with HS bound were equilibrated in the course of 400 ns MD trajectory. Sodium chlorate treatment and Heparinase III treatment To inhibit GAG sulfation, HEK293T cells were treated for 24 or 72 hours with 30mM sodium chlorate in DMEM 3% FCS. The 72-hour treatment was perfomed as an initial 48-hour treatment followed by medium renewal and a second 24-hour incubation with 30 mM sodium chlorate in DMEM3% FCS. Cells were then assessed for GP38/85 binding by flow cytometry. Heparinase III (New England Biolabs) treatment was performed according manufacturer recommendation. Briefly HEK293T cells were detached from culture support by flushing with culture medium, spun at 1000g 5min and resuspended in 20 mM Tris-HCl, 150mM NaCl, 5 mM CaCl2, 1 mM EDTA, pH 7.5 containing 30mU Heparinase III/ml. Cells were incubated 1 hour at 37°C, washed with PBS and analyzed for GP38/85 binding ability by flow cytometry. Heparin-binding analysis His-tagged purified proteins were diluted ten times in 50mM Tris pH7, 1mM NaCl, then loaded onto a Heparin column (GE Healthcare). The column was washed with 3 column volume of binding buffer and repeated elutions were performed with increasing concentration of NaCl ranging from 0.1 to 2M. Fractions were analyzed by western blot using an antiHis HRP conjugated antibody (Miltenyi Biotech). Results GP38/85 binds to cell surface Cells infected with CCHFV were shown to release GP38/85 in the supernatant [37]. In the current study, we initially analyzed its ability to bind to cell surfaces. The GP38 protein is produced from the GP85 precursor by furin cleavage between mucin protein and GP38. To produce GP85 only, we mutated lysine 246, a key basic residue of the furin cleavage site thus abrogating cleavage (Fig. 1 A) [37]. Furin cleavage sites are known to be sensistive to neighbouring O-glycans for efficient cleavage [16, 38], we therefore mutated in GP85 expressing plasmid potential O-glycosylation sites : threonine 242 and serine 245. As shown in Fig. 1 A, mutation of residue at position 242 leads to almost full GP85 precuror processing into GP38 protein, while S245A mutation did not impact GP85 cleavage. To assess cell binding capacity of these proteins, we incubated on ice, HIS tag-purified GP38 or GP85 (sequence from strain Ibar10200) with HEK293T cells and monitored the presence of cell-surface bound protein by flow cytometry using anti-His antibody. We observed a strong binding of both GP38 and GP85 to the whole cell population (Fig. 1 B). We also observed a similar binding level using a GP38/85 originating from AIGV, a virus closely related to CCHFV (Fig. 1 B). This ability to bind cell surface was identified in several tested cell lines including monocytic or lymphoid cells (THP1 and Jurkat, Fig. 1 C, and CHO Fig. 2 C). To determine the binding mechanism of GP38 to the cell surface, we treated HEK293T cells with trypsin prior to addition of GP38/85 to digest a putative cell-surface GP38/85 protein receptor. As displayed in Fig. 2 A, such treatment diminished GP38/85 binding. As trypsin cleaves cell-surface proteins, it simultaneously removes moieties associated with these proteins, including glycosaminoglycans (GAGs). These molecules, notably heparan sulfate (HS), are complex negatively charged sulfated polysaccharides with broad biological properties [35] which are widely used by viruses as cell-surface attachment sites [41, 47]. We then used CRL-2241 CHO cells, a galactosyltransferase I deficient cell line unable to perform GAG synthesis [13]. As shown in Fig. 2 B, those cells exhibited a very limited ability to bind GP38/85 in comparison to WT CHO cells. Binding of proteins to GAGs often rely on interaction of positively charged amino acids with negative charge of GAGs [22],we treated cells with sodium chlorate, a known inhibitor of GAG sulfation [36]. After 24 hours of treatment with chlorate, the binding of GP38/85 to the cell surface was reduced by 2 logs, and repeated treatment over 72 hours fully blocked GP38/85 binding (Fig. 2 C). In addition, we assayed competition with free ligands. First soluble HS was used as free competitive ligand and demonstrated a strong binding inhibition with effects seen for concentration as low as 0.1µg/mL HS (Fig. 2 D). To confirm that this interaction relies on negative charges we used a polymer of the poly(4-styrene sulfonic acid) (PSS, 75kDa form) known to be a chemical structural analog of heparin and mimicking heparin negative charges by sulfonate moieties instead sulfates ones [30]. This molecules strongly reduced GP38/85 binding to cells at concentration as low as 1nM (Fig. 2 E). This result indicates that binding of GAGs onto GP38/85 relies on negative charges. Altogether, these results identify GAGs as an efficient cell-surface attachment receptor for GP38 and GP85. GAG-binding proteins are differently sensitive to NaCl concentration depending on their affinity for thepolysaccharide [45]. To determine the strength of the GAG-GP38/85 interaction, the proteins were loaded onto a Heparin (HP) sepharose column equilibrated in a 0.1 M NaCl-containing buffer, and the proteins were eluted using a 0.1 to 2 M NaCl gradient. As shown in Fig. 2 F, GP38/85 eluted at 0.5M NaCl indicating a mid-strength interaction. In addition, all forms of GP38/85 (GP160) were bound to the column and eluted in the same fractions confirming the results of Fig. 1 B. As aforementioned, GAG binding to proteins often relies on the interaction with basic residues. These basic residues are often organized as clusters on 3D structure, or even be linearly grouped as described in the prototypic Cardin-Weintraub (C-W motif) GAG binding motif -BBXB- (where B is a basic motif amino acid and X a random amino acid) [6]. The analysis of the GP38 sequence revealed a cluster of basic residues between position 471 and 474, which fitted the Cardin-Weintraub motif (-KKNK-). This motif is conserved within all CCHFV strains including the most distant AIGV. The mapping of this motif on the atomic structure of GP38 [27], shows that these residues are not randomly distributed on the protein structure but exposed at the surface of the protein and clustered to form a defined positively charged area (Fig. 3 A-B), thus supporting a potential role as a GAG-binding domain. Basic residues of the C-W motif were mutated to glutamic acid and all mutants were produced and purified from HEK293T supernatants (Supplementary Fig. 1A). In the C-W motif, the single mutation of each of the basic residues induced a loss of GP38 ability to bind to 293T cells (Fig. 3 C). Interestingly, GP38 3D structure shows that the -BBxB- motif on the 3D structure forms with residue R412 the entry of a canyon, where the crests of both sides feature by mostly basic residues, suggesting that the C-W motif might not be solely responsible of the GAGs binding (Fig. 3 D). We then further characterized HS binding to GP38 using, in silico analysis. For this, we performed fragment-based molecular docking in “blind” mode, when a short fragment of HS (tetramer) was allowed to interact with the whole protein surface. Results revealed only one binding site, which was located in the canyon opening at the -BBxB- motif (Fig. 3 E). The tetramer in the most stable position (Fig. 3 F) showed interaction with several positively charged residues (Fig. 3 G). Then, Molecular Dynamics (MD) simulations were performed to equilibrate the complex in the presence of explicit solvent (supplementary Fig. 2A-B). For this, we used an hexameric HS fragment, which had the advantages to be as long as the binding canyon estimated by docking, and was of comparable length to many the GAG fragments length interacting with other proteins [33]. Starting from different HS orientations in respect to the protein we demonstrated the preference of SO 3 – group at N-2 position of one of N-sulfated glucosamine residues to be directed to K471 and K474 (Fig. 3 H shows the most energy favorable complex with HS, other possible binding modes are given in Supplementary Fig. 2C-F, the per-residue contacts with HS in the course of trajectory are listed in Table S1 ). The PSS used in Fig. 2 F, was also evaluated in the MD simulation and shows that this molecule predominantly occupies the GAG-binding site and interacts with the same basic residues that HS (Fig. 4 A-B). Both HS and PSS interact not only with the C-W motif but also with several other positively charged residues. In silico mutagenesis was performed and revealed that the residues K409, K396, R412, K471, K472, K474, R478, R507 strongly contribute to the HS binding (Fig. 4 C) confirming our initial findings in Fig. 3 C and expanding the number of potential interacting residues in the GAG binding domain. To confirm in silico analysis, we performed an additional set of amino acids mutations located either downstream (N394, K396, R412, K409) or upstream (R478, R507, and G509, supplementary Fig. 1B) of the -BBxB- motif amino acids and forming the binding pocket to determine their involvement in the GAGs binding ability of GP38/85 (Fig. 4 D). All but N394A impaired cell attachment phenotype (Fig. 4 E). The dual mutation of R412 and K474 that maps the entry of the canyon fully abrogated binding of GP38 to cells and therefore confirm predictions of molecular docking. Discussion GP38 function in viral cycle and its potential role in CCHFV pathology remain unclear, but the identification by Golden et al. that GP38-specific antibody can protect adult mice from lethal CCHFV challenge revealed the importance of this non-structural glycoprotein in the virus pathogenesis [15]. A more recent study has highlighted that human survivors produce protective anti-GP38 antibodies [39]. In the current study, we characterized the attachment of CCHFV GP38 to cells, and identified GAGs as cell-surface attachment receptor. We mapped the interacting domain on GP38 3D structure both using in silico prediction and mutagenesis. To produce GP38 alone, we kept initial construct containing mucin protein and we increased its processing by furin through removing inhibition due to O-glycosylation of threonine 242 residue. O-glycosylations are due to O-GalNac transferases that comprise up to 20 members that are either ubiquitously expressed or cell-type specific [4]. Future studies might investigate the nature of the O-GalNac transferase involved in T242 to better understand GP85 secretion. Indeed we demonstrated that GP85, as it contains GP38, binds to surface GAGs, which may decorate cell surface with mucin protein. GAGs are long polyanionic sulfated polysaccharide structures that are anchored on proteins present in intracellular compartments and cell surfaces [7]. In the current study, we demonstrated that GP38 binds to cell surface GAGs through different methods. Heparinase III treatment showed incomplete abrogation likely as this enzyme does not cleave all type of GAGs from the cell surface. GAG-binding domains are often composed of basic amino acids (lysine, arginine) that exhibit positive charges supposed to interact with negative charges carried by the GAGs. In our study both HS and PSS showed activity in reducing GP38 binding indicating that charge either from sulfate or sulfonate moieties are important for interaction. Of note, a very similar compound to PSS, Tolevamer has been tested in two phase II clinical trials against Clostridium difficile without significant toxicity [20], suggesting that such molecule could potentially serve as a basis for antiviral development. In GP38, the positive amino acids cluster is composed of both a prototypical Cardin-Weintraub GAG-binding domain (XBBXBX, where B are basic amino acids) and a set of surface exposed amino acids aligned on both crests of a cleft. Interestingly, among the Genbank accessible sequences of the CCHFV M segment, the prototypical binding domain xBBXBX is conserved in all sequences except one isolate from 1968 (isolate Taf,[2]). It is worth noting that one non-positively charged residue, G509, was found to be important for ligand binding. This may be due to the fact that the mutation affected the functional positioning of some basic residues, directly involved into complex formation. The experimental data from this study were fully corroborated by the in silico analysis which demonstrates that the unique docking site of HS is the one identified by our mutagenesis screen. MD experiments were run using different initial orientations of the HS fragment in respect to the protein’s binding site, and demonstrated in all tested simulations the importance of N-sulfated moieties for binding. Hundreds of proteins are known to possess GAG-binding domains that modulate their function, including proteins involved in coagulation (antithrombin), immune response (CCL21, IL-8), and growth factors/receptor signal transduction complexed (VEGF/VEGFR, FGF/FGFR). Therefore the potential activity of CCHFV GP38/85 within those pathways should be investigated to identify GP38/85 function in the viral cycle [3, 14, 19, 45]. In addition, little to no is known about the GAG biology of ticks, and therefore the role of GP38 in its natural host is even more obscure. However, several tick proteins have been described to possess GAG binding properties indicating that such motifs exist in ticks, as described for other arthropods [10, 12, 21, 32]. Previous studies have highlighted the use of monoclonal antibodies against GP38 to treat CCHFV infection, among them the BinIII protective monoclonal antibody which targets the lysine 474 residue, and therefore overlap the GAG binding domain [39]. Future investigations should define if abrogation of GAG binding by BinIII Mab is essential for its protective activity. Overall, our study provides insights into the mechanism of action of these antibodies, and sheds light on the poorly understood CCHFV GP38/85 proteins, which could improve our understanding of the viral cycle and facilitate the development of viral inhibitors. Declarations Conflict of interest Authors declare no conflict of interest. Supplemental information Supplementary Fig. 1 Author Contribution OR wrote the main manuscript text, drive the project and conduct most experiments. RV, OM provide critical reagents, protocols, performed in silico analyses and reviewed the manuscript. EG perform part of the experiments. AP, CP provide critical reagents and advice , review the manuscript. VV co - drive the project and review the manuscript. Acknowledgement We thank Ilona Novakova, Neil Kearney and Laura Cole for excellent support during their internships and Stephanie Reynard for expert technical suggestions. We thank Louis-Marie Bloyet and Mathieu Iampietro for helpful comments on the manuscript. We acknowledge FINOVI foundation for financial support and the “Investissements d’avenir” program Glyco@Alps (ANR-15-IDEX-02). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). 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Biochimie 83:811–817. doi: 10.1016/s0300-9084(01)01290-1 Suschak JJ, Golden JW, Fitzpatrick CJ, Shoemaker CJ, Badger CV, Schmaljohn CS, Garrison AR (2021) A CCHFV DNA vaccine protects against heterologous challenge and establishes GP38 as immunorelevant in mice. NPJ Vaccines 6:31. doi: 10.1038/s41541-021-00293-9 Urbinati C, Nicoli S, Giacca M, David G, Fiorentini S, Caruso A, Alfano M, Cassetta L, Presta M, Rusnati M (2009) HIV-1 Tat and heparan sulfate proteoglycan interaction: a novel mechanism of lymphocyte adhesion and migration across the endothelium. Blood 114:3335–3342. doi: 10.1182/blood-2009-01-198945 Valle-Delgado JJ, Urbán P, Fernàndez-Busquets X (2013) Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy. Nanoscale 5:3673–3680. doi: 10.1039/C2NR32821F Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J (1999) Glycosaminoglycan-binding Proteins. Cold Spring Harbor Laboratory Press Vincent MJ, Sanchez AJ, Erickson BR, Basak A, Chretien M, Seidah NG, Nichol ST (2003) Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1. J Virol 77:8640–8649. doi: 10.1128/jvi.77.16.8640-8649.2003 Vives R, Lortat-Jacob H, Fender P (2006) Heparan Sulphate Proteoglycans and Viral Vectors : Ally or Foe? CGT 6:35–44. doi: 10.2174/156652306775515565 Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25:1157–1174. doi: 10.1002/jcc.20035 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8680483","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":584593166,"identity":"bf4b359c-c9a2-4b25-8f21-cdaa72c70696","order_by":0,"name":"Olivier Reynard","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie3PsUrEMBjA8a8UWodA1y+0XF+hpZAO58Oki1l6kyAKIi0H7XI4+ySdHCIFp/oKkkPQ5RY3D0TMqUU5zOkomP+UhPzIFwCb7S/mOxXwz6078T5WgZG4WyQbCa2MZGtbjDeNJHDdBtXxLeTtgqn15VSch+3y7qQEzA2Gzp0G+XAI0TDk6eJezJpoyNKbDjCS35Okd+ZJ0XBALBkS2c8aLD1ad3CGhsHeyYsm8YrRZ9kLD8XDWhPcQWpVVJtXCAv1K9xDzpxdRP+lVvyaEyQHR2EkRaoHy/RgaCSB36qrp1M+Qb/v6EpO4/hCLB/rbt9IxgjAXvL14Cfwlq9+c8tms9n+Ya9hzk9Ysb7YlQAAAABJRU5ErkJggg==","orcid":"","institution":"CIRI, Centre International de Recherche en Infectiologie, INSERM U1111, CNRS, UMR5308, Univ Lyon, Université Claude Bernard Lyon, École Normale Supérieure de Lyon","correspondingAuthor":true,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Reynard","suffix":""},{"id":584593167,"identity":"6d2844c2-7cbf-47a0-af12-c26057f7be1f","order_by":1,"name":"Romain R. Vivès","email":"","orcid":"","institution":"Institut de Biologie Structurale","correspondingAuthor":false,"prefix":"","firstName":"Romain","middleName":"R.","lastName":"Vivès","suffix":""},{"id":584593168,"identity":"01e40f37-081d-47f3-af5c-92aafd6203ce","order_by":2,"name":"Olga Makshakova","email":"","orcid":"","institution":"Faculty of Biology, University of Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, Synthetic Biology of Signalling Processes Lab, University of Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Makshakova","suffix":""},{"id":584593169,"identity":"f56276fb-2db3-45fb-a292-f0dc99305c06","order_by":3,"name":"Estelle Gallice","email":"","orcid":"","institution":"Centre International de Recherche en Infectiologie (CIRI), INSERMU1111-CNRS UMR5308, Université Claude Bernard Lyon 1, ENS de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Estelle","middleName":"","lastName":"Gallice","suffix":""},{"id":584593170,"identity":"bac33a2c-ca57-4fbc-8315-eb157c6e662f","order_by":4,"name":"Anna Papa","email":"","orcid":"","institution":"Aristotle University of Thessaloniki","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Papa","suffix":""},{"id":584593171,"identity":"1411f854-adeb-4799-bd72-1a2397f3e359","order_by":5,"name":"Christophe Peyrefitte","email":"","orcid":"","institution":"Institut Pasteur de la Guyane","correspondingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Peyrefitte","suffix":""},{"id":584593172,"identity":"5ad95038-f096-4c53-8090-30ba5c1ee408","order_by":6,"name":"Viktor Volchkov","email":"","orcid":"","institution":"Centre International de Recherche en Infectiologie (CIRI), INSERMU1111-CNRS UMR5308, Université Claude Bernard Lyon 1, ENS de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Viktor","middleName":"","lastName":"Volchkov","suffix":""}],"badges":[],"createdAt":"2026-01-23 15:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8680483/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8680483/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101785370,"identity":"180220da-2c53-4d4c-bd1b-2eaf6fa3aed8","added_by":"auto","created_at":"2026-02-03 15:35:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGP38 and GP85 bind to cells through GAGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Analysis of GP38/85 expression profile in the supernatant of HEK293T cells transfected with 6xHis tagged GP85 expression vector (wt or T242A, S245A, K246A mutant) harvested at 48 hours post transfection. Western blot was revealed using an anti 6xHis tag HRP antibody. B) Binding of GP38 or GP85 only and GP38/85 derived from CCHFV sequence (Ibar10200) or Aigai virus GP38/85 to HEK293T after incubation on ice and subsequent analysis by flow cytometry on a BS LSR4, detection using anti His-PE. C) Binding of GP38/85 on Jurkat or THP-1 cells as described above.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/af1bd4eef775155f1538f36b.png"},{"id":101785377,"identity":"8fb09c4a-b8ab-4293-90f3-2a00633fee1e","added_by":"auto","created_at":"2026-02-03 15:35:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGP38/85 binds glycosaminoglycans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) HEK293T cells treated by 0.25 % trypsin for 5 minute at 37°C and further subjected to GP38/85 binding assay and analysed by flow cytometry. PE-mean fluorescent intensity (MFI) was normalized to the mock treated condition. B) Wildtype CHO cells or CHO cells deficient in GAG synthesis were subjected to GP38/85 binding assay, C) HEK293T cells were cultured in the presence of NaClO3 at 30 mM for 24hr or 72 hr (24hr then 48hr with medium renewal) and then subjected to GP38/85 binding assay and analysed by flow cytometry. D-E). Binding of GP38/85 on HEK293T in the presence of increasing concentration of heparan sulfate (D) or poly(4-styrene sulfonic acid) 75kDa polymer (PSS, E). F) Strength of GP38/85 – GAG interaction was evaluated through the binding of proteins to Heparin sepharose and further elution with a NaCl gradient. Fractions were analyzed by western blot and anti 6xHis HRP antibody.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/e00fa404225802d3b4b1e939.png"},{"id":101785368,"identity":"fae78153-16d2-48ec-8399-d0793430b7c8","added_by":"auto","created_at":"2026-02-03 15:35:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":697157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the GAG-binding domain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCritical amino acids for GAG-binding were mapped on the structure of GP38 (PDB 6VKF) using Chimera software. A) Cardin-Weintraub motif amino acids colored in red, B) electrostatic coloring on cell surface, Position of arginine 412 is highlighted in green. C) Individual mutants of the -BBXB- motif were produced, purified, and further assayed in cell binding assay, all proteins were assayed at 1 µg/mL. D) Position of Arginine 412 on the GP38 3D structure. E) Surface view of the most favorable binding position of a tetrameric heparan sulphate fragment determined by in silico docking. F) Detailled view of the HS fragment in the predicted binding canyon. G) The scheme of closest contacts in the GP38–heparin complex (color code: C atoms – grey for protein and green for heparin sulfate, O atoms – red, N – nitrogen, S – yellow, polar H – white, non-polar H are not shown for clarity).\u003cem\u003e H) \u003c/em\u003eMost energy favorable complex of GP38 with hexameric HS determined by molecular dynamics (MD) simulation. I) Scheme of the closest contacts in the GP38– hexameric HS complex determined by MD.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/b104df22fda103a18c88e03c.png"},{"id":101785376,"identity":"2fa93888-8fd5-453c-8aa6-aa68cb5d6bc8","added_by":"auto","created_at":"2026-02-03 15:35:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":451419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003echaracterization of the GAG binding domain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Analysis by molecular dynamics of the PSS interaction with GP38, detailled view of the PSS molecule in the predicted binding canyon. B) The scheme of closest contacts in the GP38–heparin complex (color code: C atoms – grey for protein and green for heparin sulfate, O atoms – red, N – nitrogen, S – yellow, polar H – white, non-polar H are not shown for clarity). C) Impact of the mutation of GAG binding associated residues predicted by MD. D) Critical amino acids for GAG-binding identified by structure analysis and MD were mapped on the structure of GP38. E) Individual mutants were produced, purified, and further assayed in cell binding assay, all proteins were assayed at 1 µg/mL.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/c558f42bc984bbb4d085ae0f.png"},{"id":101785378,"identity":"c0eb8fc2-9577-45dd-af6b-4a63992f1b33","added_by":"auto","created_at":"2026-02-03 15:35:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2374448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/eb9163bf-376b-4096-b1a5-5db6f2bfec6b.pdf"},{"id":101785369,"identity":"81d8d667-8f13-48d4-b80b-e3c9f8a4a7d3","added_by":"auto","created_at":"2026-02-03 15:35:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":741176,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8680483/v1/f3406af149505c844d5cd906.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CCHFV GP38 and GP85 interact with cell-surface glycosaminoglycans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCrimean Congo hemorrhagic fever virus (CCHFV) was discovered in 1944 and was identified as being the causative agent of severe hemorrhagic fever [18]. This virus belongs to the order \u003cem\u003eBunyaviridae\u003c/em\u003e, family \u003cem\u003eNairoviridae\u003c/em\u003e, genus \u003cem\u003eOrthonairovirus\u003c/em\u003e. CCHFV is an arbovirus hosted by ticks, notably those from the genus \u003cem\u003eHyalomma\u003c/em\u003e. The geographical distribution of CCHFV overlaps the distribution area of those hard ticks that ranges from Africa, Asia, central Europe, eastern Mediterranean countries (Balkan countries and, Turkey), and more recently western Europe countries e.g. Spain and France [5, 17, 34]. The first symptoms arise 5\u0026ndash;15 days after initial infection and consist of an acute febrile illness, which is followed by a severe phase generally including hemorrhagic symptoms and characterized by low platelet count, coagulopathy, acute liver infection, and the release of high amounts of pro-inflammatory mediators [5]. The disease leads to death in 5\u0026ndash;40% of the cases.\u003c/p\u003e \u003cp\u003eThe genome of CCHFV is composed of three segments termed large (L), medium (M), and,small (S) which respectively encode for the RNA polymerase, the glycoproteins and the nucleoprotein (and NS). The glycoproteins are translated as a long polyprotein of nearly 1700 amino acids that is further processed by cellular proteases, notably furin and SKI-1 proteases [37, 46]. This proteolysis and the complex O- and N- glycosylations lead to the generation of 3 predicted non-structural glycoproteins: the mucin protein, GP38, and NSm, as well as two structural glycoproteins Gn and Gc involved in low-density lipoprotein receptor (LDLR) recognition and fusion [28, 37]. The mucin protein is a highly glycosylated disordered protein that contains a high amount of O-linked glycans and five N-glycans. Mucin and GP38 proteins (also named GP1) were characterized or predicted in most nairoviruses with different levels of O-glycosylations [23]. While the mucin protein sequence is of low amino-acid homology between CCHFV isolates, GP38, Gn, NsM and Gc are more conserved [23].\u003c/p\u003e \u003cp\u003eIn the supernatant of cells transfected to express the M segment or infected with CCHFV, GP38 is mostly found in a free form, but also as a non-furin-digested precursor named GP85 containing both GP38 and the mucin protein [37]. In addition, western blot analysis of supernatants shows the presence of another, poorly characterized\u0026thinsp;~\u0026thinsp;160 kDa protein, comprising both GP38 and mucin protein [37]. Although the functional role of GP38 during CCHFV infection remains unclear, monoclonal antibodies directed against GP38 protect non-human primates from CCHFV lethal challenge and a vaccine targeting against GP38 can protect against lethal infection, thus indicating that GP38 is a critical pathogenicity factor [15, 42]. Glycosaminoglycans are targeted by a wide range of pathogens including bacteria, parasites, and viruses [1, 9, 26, 43, 44]. This observation was initially documented in 1966 in a rabbit model of HSV1 infection in which heparin was shown to protect from infection [24]. Since then, GAGs are documented as a co-factor of infection for multiple viruses, including the more recent SARS-COV-2 [11]. Here we show that secreted GP38 and GP85 bind cell surface glycosaminoglycans (GAGs) and we identify the precise binding domain.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e \u003cb\u003eCells, reagents, and transfections.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHEK293T cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium Glutamax (Thermo) supplemented with 10% fetal calf serum (FCS) and Normocin (Invivogen) at 37\u0026deg;C. Jurkat, THP1 and total lymphocytes were cultured in RPMI Glutamax (Thermo) 10% FCS. Cell transfections were performed using Jet Optimus (Polyplus) according to the manufacturer\u0026rsquo;s instructions at a ratio of 1:1 (plasmid:reagent).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmids and mutagenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe GP85 sequence was subcloned from plasmid pCDNA3 M Ibar10200 (Genbank AF467768.2) into vector phCMV with a 6xHis tag at the carboxy terminus. Aigai virus (AIGV, former CCHFV genogroup VI, AP92 strain [31]) M segment (Genbank DQ211625.1) was cloned by RT-PCR from RNA extracted from the supernatant of infected cells and GP85 was further subcloned into phCMV vector with a 6xHis tag at the carboxy terminus. Mutagenesis of GP38 plasmid was performed using the Q5 mutagenesis kit (New England Biolabs) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein production and purification\u003c/h2\u003e \u003cp\u003eCulture medium from phCMV GP85-6xHIS transfected HEK 293Tcells was harvested 48 to 72 hours post-transfection, clarified by centrifugation (3000g, 5 minutes), and filtered through 0.22\u0026micro;m filter (Millipore). Tetradentate chelating agarose resin with divalent cobalt (Co2+) (Thermo) was used for the affinity purification step. Binding was performed in the presence of 5 mM imidazole. The resin was washed twice with a 30-bed volume of PBS 10mM Imidazole. Elution was performed by applying a 200 mM Imidazole solution in PBS pH 7.5. The proteins were further dialyzed against PBS in 5kDa cartridge (Thermo). The purified proteins contain an average of 85% of GP38, 12% of GP85 (precursor), and 3% of GP160.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow cytometry analysis of GP38/85 binding\u003c/h3\u003e\n\u003cp\u003eBinding assay was perfomed on 10\u003csup\u003e5\u003c/sup\u003e cells per condition that were incubated on ice for 5 minutes, then spun for 1 minute at 750g and incubated with purified GP38/85 proteins (10\u0026micro;g/mL) for 30 minutes on ice. Cells were then washed twice with ice-cold PBS and stained using an anti 6xHIS antibody labeled with phycoerythrin (Miltenyi biotech) for 20 minutes on ice. Stained cells were washed twice with ice-cold PBS and analyzed directly on an LSR Fortessa 5L flow cytometer (Becton Dickinson).\u003c/p\u003e\n\u003ch3\u003eIn silico simulations\u003c/h3\u003e\n\u003cp\u003eTo identify the location of heparin-binding site, the molecular docking of heparin to GP38 was performed using ClusPro web server [29]. Then, complexes of GP38 and heparin were equilibrated using molecular dynamics simulations to take explicitly into account both ligand and protein flexibility in water solvent. The hexamer of heparan sulfate was placed in a proximity of the binding site, determined by molecular docking, and at the distance ~\u0026thinsp;5\u0026Aring; from the protein surface to allow both protein and polysaccharide to adjust their geometries in the course of the complex formation. Molecular dynamics simulations were carried out in Amber 18 package[8], GLYCAM06j force field was used for heparin [40], generalized amber force field (gaff) for the chemical analog of GAG [48], and AMBER14SB for protein molecule [25]. The in silico site-directed mutagenesis was performed based on above described structures of native GP38 and HS complexes via a replacement of a target basic residue by glutamic acid. The new structures GP38 mutants with HS bound were equilibrated in the course of 400 ns MD trajectory.\u003c/p\u003e\n\u003ch3\u003eSodium chlorate treatment and Heparinase III treatment\u003c/h3\u003e\n\u003cp\u003eTo inhibit GAG sulfation, HEK293T cells were treated for 24 or 72 hours with 30mM sodium chlorate in DMEM 3% FCS. The 72-hour treatment was perfomed as an initial 48-hour treatment followed by medium renewal and a second 24-hour incubation with 30 mM sodium chlorate in DMEM3% FCS. Cells were then assessed for GP38/85 binding by flow cytometry. Heparinase III (New England Biolabs) treatment was performed according manufacturer recommendation. Briefly HEK293T cells were detached from culture support by flushing with culture medium, spun at 1000g 5min and resuspended in 20 mM Tris-HCl, 150mM NaCl, 5 mM CaCl2, 1 mM EDTA, pH 7.5 containing 30mU Heparinase III/ml. Cells were incubated 1 hour at 37\u0026deg;C, washed with PBS and analyzed for GP38/85 binding ability by flow cytometry.\u003c/p\u003e\n\u003ch3\u003eHeparin-binding analysis\u003c/h3\u003e\n\u003cp\u003eHis-tagged purified proteins were diluted ten times in 50mM Tris pH7, 1mM NaCl, then loaded onto a Heparin column (GE Healthcare). The column was washed with 3 column volume of binding buffer and repeated elutions were performed with increasing concentration of NaCl ranging from 0.1 to 2M. Fractions were analyzed by western blot using an antiHis HRP conjugated antibody (Miltenyi Biotech).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGP38/85 binds to cell surface\u003c/h2\u003e \u003cp\u003eCells infected with CCHFV were shown to release GP38/85 in the supernatant [37]. In the current study, we initially analyzed its ability to bind to cell surfaces. The GP38 protein is produced from the GP85 precursor by furin cleavage between mucin protein and GP38. To produce GP85 only, we mutated lysine 246, a key basic residue of the furin cleavage site thus abrogating cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [37]. Furin cleavage sites are known to be sensistive to neighbouring O-glycans for efficient cleavage [16, 38], we therefore mutated in GP85 expressing plasmid potential O-glycosylation sites : threonine 242 and serine 245. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, mutation of residue at position 242 leads to almost full GP85 precuror processing into GP38 protein, while S245A mutation did not impact GP85 cleavage. To assess cell binding capacity of these proteins, we incubated on ice, HIS tag-purified GP38 or GP85 (sequence from strain Ibar10200) with HEK293T cells and monitored the presence of cell-surface bound protein by flow cytometry using anti-His antibody. We observed a strong binding of both GP38 and GP85 to the whole cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We also observed a similar binding level using a GP38/85 originating from AIGV, a virus closely related to CCHFV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This ability to bind cell surface was identified in several tested cell lines including monocytic or lymphoid cells (THP1 and Jurkat, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, and CHO Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the binding mechanism of GP38 to the cell surface, we treated HEK293T cells with trypsin prior to addition of GP38/85 to digest a putative cell-surface GP38/85 protein receptor. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, such treatment diminished GP38/85 binding. As trypsin cleaves cell-surface proteins, it simultaneously removes moieties associated with these proteins, including glycosaminoglycans (GAGs). These molecules, notably heparan sulfate (HS), are complex negatively charged sulfated polysaccharides with broad biological properties [35] which are widely used by viruses as cell-surface attachment sites [41, 47]. We then used CRL-2241 CHO cells, a galactosyltransferase I deficient cell line unable to perform GAG synthesis [13]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, those cells exhibited a very limited ability to bind GP38/85 in comparison to WT CHO cells. Binding of proteins to GAGs often rely on interaction of positively charged amino acids with negative charge of GAGs [22],we treated cells with sodium chlorate, a known inhibitor of GAG sulfation [36]. After 24 hours of treatment with chlorate, the binding of GP38/85 to the cell surface was reduced by 2 logs, and repeated treatment over 72 hours fully blocked GP38/85 binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, we assayed competition with free ligands. First soluble HS was used as free competitive ligand and demonstrated a strong binding inhibition with effects seen for concentration as low as 0.1\u0026micro;g/mL HS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To confirm that this interaction relies on negative charges we used a polymer of the poly(4-styrene sulfonic acid) (PSS, 75kDa form) known to be a chemical structural analog of heparin and mimicking heparin negative charges by sulfonate moieties instead sulfates ones [30]. This molecules strongly reduced GP38/85 binding to cells at concentration as low as 1nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This result indicates that binding of GAGs onto GP38/85 relies on negative charges. Altogether, these results identify GAGs as an efficient cell-surface attachment receptor for GP38 and GP85. GAG-binding proteins are differently sensitive to NaCl concentration depending on their affinity for thepolysaccharide [45]. To determine the strength of the GAG-GP38/85 interaction, the proteins were loaded onto a Heparin (HP) sepharose column equilibrated in a 0.1 M NaCl-containing buffer, and the proteins were eluted using a 0.1 to 2 M NaCl gradient. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, GP38/85 eluted at 0.5M NaCl indicating a mid-strength interaction. In addition, all forms of GP38/85 (GP160) were bound to the column and eluted in the same fractions confirming the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eAs aforementioned, GAG binding to proteins often relies on the interaction with basic residues. These basic residues are often organized as clusters on 3D structure, or even be linearly grouped as described in the prototypic Cardin-Weintraub (C-W motif) GAG binding motif -BBXB- (where B is a basic motif amino acid and X a random amino acid) [6]. The analysis of the GP38 sequence revealed a cluster of basic residues between position 471 and 474, which fitted the Cardin-Weintraub motif (-KKNK-). This motif is conserved within all CCHFV strains including the most distant AIGV. The mapping of this motif on the atomic structure of GP38 [27], shows that these residues are not randomly distributed on the protein structure but exposed at the surface of the protein and clustered to form a defined positively charged area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B), thus supporting a potential role as a GAG-binding domain. Basic residues of the C-W motif were mutated to glutamic acid and all mutants were produced and purified from HEK293T supernatants (Supplementary Fig.\u0026nbsp;1A). In the C-W motif, the single mutation of each of the basic residues induced a loss of GP38 ability to bind to 293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, GP38 3D structure shows that the -BBxB- motif on the 3D structure forms with residue R412 the entry of a canyon, where the crests of both sides feature by mostly basic residues, suggesting that the C-W motif might not be solely responsible of the GAGs binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We then further characterized HS binding to GP38 using, \u003cem\u003ein silico\u003c/em\u003e analysis. For this, we performed fragment-based molecular docking in \u0026ldquo;blind\u0026rdquo; mode, when a short fragment of HS (tetramer) was allowed to interact with the whole protein surface. Results revealed only one binding site, which was located in the canyon opening at the -BBxB- motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The tetramer in the most stable position (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) showed interaction with several positively charged residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Then, Molecular Dynamics (MD) simulations were performed to equilibrate the complex in the presence of explicit solvent (supplementary Fig.\u0026nbsp;2A-B). For this, we used an hexameric HS fragment, which had the advantages to be as long as the binding canyon estimated by docking, and was of comparable length to many the GAG fragments length interacting with other proteins [33]. Starting from different HS orientations in respect to the protein we demonstrated the preference of SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e group at N-2 position of one of N-sulfated glucosamine residues to be directed to K471 and K474 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH shows the most energy favorable complex with HS, other possible binding modes are given in Supplementary Fig.\u0026nbsp;2C-F, the per-residue contacts with HS in the course of trajectory are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The PSS used in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, was also evaluated in the MD simulation and shows that this molecule predominantly occupies the GAG-binding site and interacts with the same basic residues that HS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Both HS and PSS interact not only with the C-W motif but also with several other positively charged residues. \u003cem\u003eIn silico\u003c/em\u003e mutagenesis was performed and revealed that the residues K409, K396, R412, K471, K472, K474, R478, R507 strongly contribute to the HS binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) confirming our initial findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and expanding the number of potential interacting residues in the GAG binding domain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm \u003cem\u003ein silico\u003c/em\u003e analysis, we performed an additional set of amino acids mutations located either downstream (N394, K396, R412, K409) or upstream (R478, R507, and G509, supplementary Fig.\u0026nbsp;1B) of the -BBxB- motif amino acids and forming the binding pocket to determine their involvement in the GAGs binding ability of GP38/85 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). All but N394A impaired cell attachment phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The dual mutation of R412 and K474 that maps the entry of the canyon fully abrogated binding of GP38 to cells and therefore confirm predictions of molecular docking.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGP38 function in viral cycle and its potential role in CCHFV pathology remain unclear, but the identification by Golden \u003cem\u003eet al.\u003c/em\u003e that GP38-specific antibody can protect adult mice from lethal CCHFV challenge revealed the importance of this non-structural glycoprotein in the virus pathogenesis [15]. A more recent study has highlighted that human survivors produce protective anti-GP38 antibodies [39]. In the current study, we characterized the attachment of CCHFV GP38 to cells, and identified GAGs as cell-surface attachment receptor. We mapped the interacting domain on GP38 3D structure both using in silico prediction and mutagenesis. To produce GP38 alone, we kept initial construct containing mucin protein and we increased its processing by furin through removing inhibition due to O-glycosylation of threonine 242 residue. O-glycosylations are due to O-GalNac transferases that comprise up to 20 members that are either ubiquitously expressed or cell-type specific [4]. Future studies might investigate the nature of the O-GalNac transferase involved in T242 to better understand GP85 secretion. Indeed we demonstrated that GP85, as it contains GP38, binds to surface GAGs, which may decorate cell surface with mucin protein. GAGs are long polyanionic sulfated polysaccharide structures that are anchored on proteins present in intracellular compartments and cell surfaces [7]. In the current study, we demonstrated that GP38 binds to cell surface GAGs through different methods. Heparinase III treatment showed incomplete abrogation likely as this enzyme does not cleave all type of GAGs from the cell surface. GAG-binding domains are often composed of basic amino acids (lysine, arginine) that exhibit positive charges supposed to interact with negative charges carried by the GAGs. In our study both HS and PSS showed activity in reducing GP38 binding indicating that charge either from sulfate or sulfonate moieties are important for interaction. Of note, a very similar compound to PSS, Tolevamer has been tested in two phase II clinical trials against \u003cem\u003eClostridium difficile\u003c/em\u003e without significant toxicity [20], suggesting that such molecule could potentially serve as a basis for antiviral development. In GP38, the positive amino acids cluster is composed of both a prototypical Cardin-Weintraub GAG-binding domain (XBBXBX, where B are basic amino acids) and a set of surface exposed amino acids aligned on both crests of a cleft. Interestingly, among the Genbank accessible sequences of the CCHFV M segment, the prototypical binding domain xBBXBX is conserved in all sequences except one isolate from 1968 (isolate Taf,[2]). It is worth noting that one non-positively charged residue, G509, was found to be important for ligand binding. This may be due to the fact that the mutation affected the functional positioning of some basic residues, directly involved into complex formation. The experimental data from this study were fully corroborated by the \u003cem\u003ein silico\u003c/em\u003e analysis which demonstrates that the unique docking site of HS is the one identified by our mutagenesis screen. MD experiments were run using different initial orientations of the HS fragment in respect to the protein\u0026rsquo;s binding site, and demonstrated in all tested simulations the importance of N-sulfated moieties for binding. Hundreds of proteins are known to possess GAG-binding domains that modulate their function, including proteins involved in coagulation (antithrombin), immune response (CCL21, IL-8), and growth factors/receptor signal transduction complexed (VEGF/VEGFR, FGF/FGFR). Therefore the potential activity of CCHFV GP38/85 within those pathways should be investigated to identify GP38/85 function in the viral cycle [3, 14, 19, 45]. In addition, little to no is known about the GAG biology of ticks, and therefore the role of GP38 in its natural host is even more obscure. However, several tick proteins have been described to possess GAG binding properties indicating that such motifs exist in ticks, as described for other arthropods [10, 12, 21, 32]. Previous studies have highlighted the use of monoclonal antibodies against GP38 to treat CCHFV infection, among them the BinIII protective monoclonal antibody which targets the lysine 474 residue, and therefore overlap the GAG binding domain [39]. Future investigations should define if abrogation of GAG binding by BinIII Mab is essential for its protective activity. Overall, our study provides insights into the mechanism of action of these antibodies, and sheds light on the poorly understood CCHFV GP38/85 proteins, which could improve our understanding of the viral cycle and facilitate the development of viral inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eAuthors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eSupplemental information\u003c/h2\u003e \u003cp\u003eSupplementary Fig.\u0026nbsp;1\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOR wrote the main manuscript text, drive the project and conduct most experiments. RV, OM provide critical reagents, protocols, performed in silico analyses and reviewed the manuscript. EG perform part of the experiments. AP, CP provide critical reagents and advice , review the manuscript. VV co - drive the project and review the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Ilona Novakova, Neil Kearney and Laura Cole for excellent support during their internships and Stephanie Reynard for expert technical suggestions. We thank Louis-Marie Bloyet and Mathieu Iampietro for helpful comments on the manuscript. We acknowledge FINOVI foundation for financial support and the \u0026ldquo;Investissements d\u0026rsquo;avenir\u0026rdquo; program Glyco@Alps (ANR-15-IDEX-02). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltg\u0026auml;rde N, Eriksson C, Peerboom N, Phan-Xuan T, Moeller S, Schnabelrauch M, Svedhem S, Trybala E, Bergstr\u0026ouml;m T, Bally M (2015) Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction. 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J Comput Chem 25:1157\u0026ndash;1174. doi: 10.1002/jcc.20035\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8680483/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8680483/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCrimean Congo Hemorrhagic Fever Virus (CCHFV) is a negative-strand segmented RNA virus responsible for severe hemorrhagic fever in humans. The M genomic segment of CCHFV encodes a polyprotein precursor that is processed by cellular proteases into several structural and non-structural proteins. Among them, GP38 and its precursor GP85 are known to be secreted into the extracellular environment. We investigated their abilities to bind cells and we identified that they strongly bind to the cell plasma membrane through interaction with glycosaminoglycans. This interaction was mapped to a surface-exposed basic cluster that combines both a prototypical GAG-binding domain and linearly distant amino-acids. The present study describes for the first time the CCHFV GP38/85 proteins interaction with glycosaminoglycan and characterizes the interaction domain.\u003c/p\u003e","manuscriptTitle":"CCHFV GP38 and GP85 interact with cell-surface glycosaminoglycans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 15:35:24","doi":"10.21203/rs.3.rs-8680483/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T15:15:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T14:49:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-06T13:38:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-06T11:10:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197547678989830556361447529383570358710","date":"2026-02-04T04:13:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321828036394851224150893801042636757366","date":"2026-02-03T16:30:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152114748567437037220211904224736829483","date":"2026-02-02T20:12:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111631980533101095647599035865240300770","date":"2026-02-02T09:08:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T08:47:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T07:55:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-29T03:57:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Viruses","date":"2026-01-23T14:50:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"57a0fa8e-6a94-454c-b8e8-0379f27001d4","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62190856,"name":"Biological sciences/Biochemistry"},{"id":62190857,"name":"Biological sciences/Microbiology"},{"id":62190858,"name":"Biological sciences/Molecular biology"},{"id":62190859,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2026-05-07T16:09:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 15:35:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8680483","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8680483","identity":"rs-8680483","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}