The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection (original) (raw)

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Division of Molecular Pathobiology, Hokkaido University, Sapporo

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Division of Molecular Pathobiology, Hokkaido University, Sapporo

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Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Japan

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Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Japan

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Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo

National Virus Reference Laboratory, School of Medicine, University College Dublin, Ireland

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Laboratory of Biomolecular Science, Faculty of Pharmaceutical Science, Hokkaido University, Sapporo

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Laboratory of Biomolecular Science, Faculty of Pharmaceutical Science, Hokkaido University, Sapporo

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Laboratory of Biomolecular Science, Faculty of Pharmaceutical Science, Hokkaido University, Sapporo

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Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo

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Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo

Center for Research in Infectious Diseases, University College Dublin, Ireland

Global Virus Network, Baltimore, Maryland

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Present address: Toyoyuki Ose, Laboratory of X-Ray Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.

Author Notes

Received:

06 November 2017

Accepted:

23 February 2018

Published:

26 February 2018

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Michihito Sasaki, Paulina D Anindita, Naoto Ito, Makoto Sugiyama, Michael Carr, Hideo Fukuhara, Toyoyuki Ose, Katsumi Maenaka, Ayato Takada, William W Hall, Yasuko Orba, Hirofumi Sawa, The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection, The Journal of Infectious Diseases, Volume 217, Issue 11, 1 June 2018, Pages 1740–1749, https://doi.org/10.1093/infdis/jiy081
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Abstract

Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS _N_- and 6-_O_-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV–HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.

Rabies virus (RABV), genus Lyssavirus, family Rhabdoviridae, is the causative agent of rabies, a fatal neurological disease in mammals, including humans. For establishment of RABV infection, the virion attaches to target cells and binds to an RABV-specific receptor molecule, followed by cellular internalization by endocytosis [1, 2]. Rabies virus uses several host membrane molecules as entry receptors, including the nicotinic acetylcholine receptor, neural cell adhesion molecule (NCAM), and nerve growth factor receptor p75NTR [3–5]. Although characterization of the interaction of RABV with these receptors has facilitated our understanding of RABV infection, the mechanism of cellular attachment and entry of RABV remains to be elucidated.

As with other viruses, cellular attachment is an initial step in RABV infection. Viral attachment factors are host molecules that facilitate viral adhesion on the cell surface and enhance infection but do not trigger endocytic entry and membrane fusion of viruses. The glycoprotein (G) of RABV, the only transmembrane protein of RABV, binds to the entry receptors and plays critical roles during RABV entry [1, 2]. Rabies virus G also binds various cell membrane components, such as membranous proteins, gangliosides, and lipids [6–9]. These studies suggested the existence of the attachment factor(s) that contributes to efficient adhesion of RABV; however, the molecules that serve as attachment factors for RABV infection are unidentified, and the mechanism of cellular attachment of RABV remains unclear.

Heparan sulfate (HS), a glycosaminoglycan with a disaccharide repeating unit of glucosamine and uronic acid, is abundant on cellular surfaces as a component of HS proteoglycans. Heparan sulfate is involved in various biological events through interactions with cytokines, growth factors, proteases, and membrane proteins [10]. Previous work has shown that HS also binds certain viruses and mediates target cell infection [11–21]. Soluble forms of HS and heparin, a highly sulfated HS analog, can neutralize infection with viruses that use HS for cellular attachment by competitive inhibition for binding of cellular HS to the viruses [12–21]. Heparan sulfate and heparin have also been shown to inhibit RABV entry; however, this observation has been explained by the assumption that HS antagonizes the binding of RABV to the entry receptor NCAM because HS also binds to NCAM [4]. Thus, the tripartite relationship between RABV, HS, and NCAM is still unclear. In this study, we investigated the role of cellular HS on RABV infection and examined the interaction between HS and RABV.

METHODS

Cells and Viruses

SK-N-SH and NA cells were maintained in Eagle’s minimal essential medium (MEM) with 10% fetal bovine serum (FBS). A549 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS. Huh-7 cells were maintained in Roswell Park Memorial Insitute 1640 medium with 10% FBS. The master stock of the RABV challenge virus standard (CVS) strain was prepared by intracranial infection of suckling mice. The master stock of RABV high egg passage Flury (HEP) strain was propagated in NA cells. Working stocks of the RABV strains were propagated in NA cells once and titrated by focus-forming assays on NA cells as described previously [22].

Quantification of Infectivity

SK-N-SH and Huh-7 cells were infected with RABV at a multiplicity of infection (MOI) of 5. A549 cells were infected with RABV at a MOI of 25. The indicated MOIs enable evaluation for the effect of treatments. Following incubation for 1 hour, cells were washed with phosphate-buffered saline (PBS) and cultured for 18 hours. Cells were fixed with 3.7% buffered formaldehyde, permeabilized with PBS/0.2% Triton X-100, and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-RABV monoclonal antibody (800-092, Fujirebio Diagnostics) and Hoechst 33342 (Molecular Probes). Images were captured with an automated microscope IN Cell Analyzer 2000 (GE Healthcare), and infection rates were calculated by counting total cells and RABV-positive cells.

Enzymatic Removal of Cellular HS

Cells were treated with heparinase III (Sigma-Aldrich) in serum-free DMEM at 37°C for 1 hour. After PBS washing, cells were infected with RABV. Viral infectivity was quantified as described above.

Virus Infection Assay With Heparan Sulfate

For evaluation of the effect of viral treatment with HS, RABV was treated with a range of dilutions of heparin (Wako), HS (Iduron), 2-_O_-desulfated heparin (Iduron), 6-_O_-desulfated heparin (Iduron), or de-_N_-sulfated heparin (Sigma-Aldrich) for 1 hour, then inoculated to cells. Following incubation for 1 hour, cells were washed with PBS and cultured for 18 hours. For evaluation of the effect of cellular treatment with HS or heparin, cells were treated with heparin at 37°C for 1 hour, thoroughly washed with PBS twice, and infected with RABV. Viral infectivity was quantified as described above.

Pull-Down Assays

Rabies virus and heparin sepharose (Abcam) were incubated in 0.1 M phosphate buffer (PB; pH 7.0) with/without 300 μg/mL of heparin at 4°C for 1 hour. Glutathione sepharose 4B (GE Healthcare) was used as control. After PB washing, proteins were eluted and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SAS-PAGE) and detected by immunoblotting with anti-RABV N (3R7-5B12, HyTest).

Preparation of a Recombinant Glycoprotein and Surface Plasmon Resonance Analysis

A recombinant trimeric form of CVS-G (rG) was prepared by baculovirus expression (Invitrogen). Surface plasmon resonance (SPR) measurements were performed using Biacore 3000 (GE Healthcare) as described previously [20]. Further information is detailed in the Supplementary Methods.

Gene Knockdown

Three small interfering RNAs (siRNAs) targeting each gene (Silencer Select predesigned siRNA, Ambion) were examined independently. Silencer Select negative control siRNA was used as a control. Cells were reverse-transfected with 20 nM of siRNA using Lipofectamine RNAiMAX (Invitrogen) in 96-well plates. Following culture for 68 hours, cells were infected with RABV. Viral infectivity was quantified as described above. Cell toxicities of the gene knockdowns were assessed with the CellTiter-Glo assay (Promega).

Pseudotyped Vesicular Stomatitis Virus Infection Assay

Rabies virus G genes were cloned into the pCXSN [23] or pCI (Promega) vectors. The HEP/CVS chimeric G-expressing plasmids were generated by exchanging partial regions of HEP-G with the homologous region of CVS-G using an In-Fusion HD Cloning kit (Clontech). G mutants were generated by site-directed mutagenesis of plasmids. Replication-incompetent vesicular stomatitis virus (VSV) containing the green fluorescent protein (GFP) gene (VSVΔG*) was generated as described previously [24]. VSVΔG* pseudotyped with RABV G or G mutants were treated with/without 100 μg/mL of heparin at 37°C for 30 minutes and inoculated to SK-N-SH. Infectivity was calculated by counting nuclei stained with Hoechst 33342 and GFP-expressing cells with IN Cell Analyzer 2000 (GE Healthcare).

Statistical Analysis

One-way analysis of variance (ANOVA) with Dunnett’s test was used to determine statistical significance.

RESULTS

Neutralizing Activity of Soluble Heparan Sulfate Against Rabies Virus Infection in Neuronal and Nonneuronal Cells

Using a RABV CVS strain, we initially assessed the anti-RABV activity of soluble HS upon infection of neuronal SK-N-SH cells expressing NCAM (Figure 1A). Preincubation of RABV with soluble HS neutralized viral infectivity in a dose-dependent manner (Figure 1B), and heparin exhibited a stronger anti-RABV effect than HS (Figure 1C). Preincubation of SK-N-SH cells with HS or heparin, followed by PBS washing to remove unbound HS and heparin, showed no effect on RABV infectivity (Figure 1D and 1E). These results suggested that the neutralization activities of soluble HS and heparin on RABV infectivity are related to an interaction with RABV rather than NCAM on cells.

Figure 1.

Antiviral effect of exogenous heparan sulfate (HS) and heparin on the infectivity of rabies virus (RABV) challenge virus standard (CVS) strain. A, Cellular surface expression level of neural cell adhesion molecule (NCAM) was confirmed by flow cytometry using anti-NCAM antibody. Control mouse immunoglobulin G2b antibody was used as a staining control. B and C, The RABV CVS was mixed with the indicated concentrations of HS (B) and heparin (C), and then inoculated to SK-N-SH cells. Rabies virus–infected cells were identified by indirect immunofluorescence with anti-RABV N antibody and Hoechst 33342 and counted by imaging cytometry. D and E, The SK-N-SH cells were incubated with HS (D) or heparin (E) and repeatedly washed with phosphate-buffered saline prior to infection with RABV CVS. Rabies virus–infected cells were analyzed as described above. F_–_I, The same experiments (B_–_E) were performed using Huh-7 cells. The values in the graphs are shown as means ± standard deviations of triplicates from a representative experiment. Statistical analyses were performed using one-way analysis of variance with Dunnett’s test. * P < .01, significantly different from the control. Abbreviations: IgG2b, immunoglobulin G2b; NCAM, neural cell adhesion molecule.

To further clarify the anti-RABV activity of soluble HS and heparin, we performed experiments using NCAM-negative, nonneuronal Huh-7 cells (Figure 1A). As observed in SK-N-SH cells, preincubation of RABV CVS with either HS or heparin neutralized viral infectivity in a dose-dependent manner, and heparin had a strong anti-RABV effect (Figure 1F and 1G). In addition, preincubation of Huh-7 cells with HS or heparin did not decrease RABV infectivity (Figure 1H and 1I). Comparable results were also obtained in experiments using another nonneuronal cell, A549 (Supplementary Figure 1, A–E). Taken together, these findings indicate that exogenous soluble HS or heparin neutralizes RABV infection independent of cellular NCAM.

Inhibition of Rabies Virus Infection by Enzymatic Depletion of Cellular Heparan Sulfate

To further examine the role of cellular HS proteoglycans in RABV infection, we inoculated SK-N-SH cells with RABV CVS after enzymatic depletion of cellular HS. Treatment with heparinase III reduced surface HS of SK-N-SH cells (Figure 2A) and reduced the cellular susceptibility to RABV infection in a dose-dependent manner (Figure 2B and 2C). Similar to SK-N-SH, treatment with heparinase III inhibited RABV infection in both Huh-7 (Figure 2D) and A549 cells (Supplementary Figure 1F). These results indicate that cellular HS is required for the efficient RABV infection of both neuronal and nonneuronal cells.

Figure 2.

Effect of enzymatic removal of cellular heparan sulfate (HS) on the infectivity of rabies virus (RABV) challenge virus standard (CVS). A, SK-N-SH cells were treated with the indicated concentrations of heparinase III and then analyzed by flow cytometry with anti-HS immunobulin M (IgM) antibody. Control mouse IgM was used as control antibody. B and C, The SK-N-SH cells treated with heparinase III were infected with RABV CVS. The cells were stained with anti-RABV N antibody (green) and Hoechst 33342 nuclear dye (blue). The infected cells were subjected to fluorescent microscopy analysis (B) and counted by imaging cytometry (C). Scale bars, 100 μm. D, Huh-7 cells were pretreated with heparinase III and then infected with RABV CVS. Cells were stained and counted by imaging cytometry as described above. The values in the graphs are shown as means ± standard deviations of triplicates from a representative experiment. Statistical analyses were performed using one-way analysis of variance with Dunnett’s test. * P < .01, significantly different from the control.

Direct Binding of Rabies Virus and Its Glycoprotein to Heparin

Because preincubation of RABV with heparin, but not cells, inhibited viral infectivity, we assessed whether there was a physical interaction between heparin and RABV. Pull-down experiments with RABV CVS virions and heparin-conjugated sepharose beads were performed. Rabies virus specifically bound to heparin-conjugated beads, and this interaction was reduced by addition of heparin to the mixture of RABV and heparin-conjugated beads (Figure 3A). To elucidate the anti-RABV mechanism of heparin, we examined the cellular attachment of RABV in the presence of heparin. After incubation of RABV virions with cells at 4°C, attached virions were detected by flow cytometry. Pretreatment of virions with heparin markedly reduced viral attachment on the cell surface (Figure 3B). These results indicate that heparin can directly bind to RABV virions and that this interaction decreases viral attachment on the target cell surface.

Figure 3.

Direct binding of heparin to rabies virus (RABV) challenge virus standard (CVS). A, Control or heparin sepharose beads were incubated with RABV CVS in phosphate-buffered saline. Heparin was used to compete the binding of RABV CVS to beads. The viruses binding to the beads were detected by immunoblot with anti-RABV N protein. B, Rabies virus CVS and detached cells were incubated in the absence or presence of heparin at 4°C. The attached viruses on the cell surface were detected by flow cytometry with anti-RABV G protein. Abbreviations: CVS, challenge virus standard; MOI, multiplicity of infection; RABV, rabies virus.

Among the 5 structural proteins of RABV, G is the major protein distributed on the viral envelope in a homotrimeric structure [25]. Therefore, we hypothesized that G is a possible ligand for HS. To analyze the interaction of G derived from RABV CVS (CVS-G) and HS, we generated a recombinant trimeric form of CVS-G (rG) containing the ectodomain of CVS-G and the foldon trimerization domain [26]. We first confirmed that rG bound to cells (red line in Figure 4A) and the binding was inhibited in the presence of heparin (Figure 4A). The rG also neutralized the infectivity of RABV CVS, suggesting that rG possesses a domain to attach to the cell surface, competiting with RABV (Supplementary Figure 2). We analyzed rG binding to heparin by SPR assay. When rG was injected onto the heparin-immobilized sensor chip, a binding response signal was observed, and this increased in a dose-dependent manner (Figure 4B). These results indicate that rG plays a role as the ligand for heparin.

Figure 4.

Direct binding of heparin to rabies virus (RABV) glycoprotein G. A, Recombinant soluble challenge virus standard G (CVS-G) protein was incubated with SK-N-SH cells at 4°C. The binding of G protein was detected by flow cytometry with anti-RABV G protein. B, Sensorgrams of the binding of recombinant CVS-G protein (rG) to heparin immobilized on a sensor chip by surface plasmon resonance analysis. Abbreviation: RABV, rabies virus; rG, recombinant challenge virus standard G protein; RU, response units.

Contribution of Type _N_- and 6-_O_-Sulfations on Cellular Heparan Sulfate to Rabies Virus Infection

During HS biosynthesis, HS chains are modified by a series of sulfations: _N_-, 2-_O_-, 3-_O_- and 6-_O_-sulfations. The sulfation pattern of HS is dependent on cell type and modifies ligand-binding properties [10]. To analyze the contribution of these sulfate modifications on interactions between HS and RABV, we examined the neutralizing activity of desulfated heparins on RABV CVS infection. Heparins selectively desulfated in _N_-sulfation (DeN), 2-_O_-sulfation (De2O), and 6-_O_-sulfation (De6O) were used. The viral neutralizing activity of De2O-sulfated heparin was comparable with that of native heparin, whereas DeN- and De6O-sulfated heparins had no effect on viral infection at the examined concentrations (Figure 5A). Using desulfated heparins, we estimated the interaction between each of the desulfated heparins and rG by competition binding assays using SPR. Addition of either heparin or De2O-sulfated heparin decreased the binding of rG to heparin in a dose-dependent manner, whereas DeN- or De6O-sulfated heparins showed less inhibitory effect on the binding of rG to heparin (Figure 5B), indicating that heparin and De2O-sulfated heparin, but not DeN-sulfated heparin and De6O-sulfated heparin, efficiently interacted with rG and interfered with the binding of rG to heparin. These data suggest that _N_- and 6-_O_-sulfations of heparin are required for the binding to rG and contribute to the antiviral neutralizing activity for RABV infection.

Figure 5.

Contribution of sulfation types of heparin and heparan sulfate (HS) for their interaction with rabies virus (RABV) challenge virus standard (CVS). A, Rabies virus CVS was mixed with either heparin or the indicated forms of desulfated heparin and then inoculated to SK-N-SH cells. Cells were stained with anti-RABV N antibody and Hoechst 33342 and counted by imaging cytometry. Relative infectivity was calculated by setting the infectivity of mock-treated RABV CVS to 100%. B, Sensorgrams of the binding of recombinant CVS-G protein (rG) to heparin immobilized on a sensor chip, after preincubation with heparin or the indicated forms of desulfated heparin. C, Rabies virus CVS was inoculated with SK-N-SH cells transfected with small interfering RNAs (siRNAs) targeting the indicated genes. Three siRNAs were assayed for each gene knockdown. Cells were analyzed by imaging cytometry, and relative infectivity was calculated as described above. The values in the graphs are shown as means ± standard deviations of triplicates from a representative experiment. Statistical analyses were performed using one-way analysis of variance with Dunnett’s test. * P < .01, significantly different from the control. Abbreviation: RU, response units.

We examined the effect of sulfate modifications of cellular HS on RABV infectivity. Four isoforms of _N_-deacetylase/_N_-sulfotransferase (encoded by _NDST1_-4), 2-_O_-sulfotransferase (HS2ST1), and 3 isoforms of 6-_O_-sulfotransferase (HS6ST1-3) have been identified as host enzymes catalyzing the reactions of _N_-, 2-_O_-, and 6-_O_-sulfations in cellular HS biosynthesis, respectively [10]. Endogenous expression of the 8 genes in SK-N-SH was confirmed by quantitative reverse transcription PCR (RT-qPCR) (Supplementary Figure 3). At least 2 of 3 tested siRNAs that targeted NDST1, NDST2, NDST3, and HS6ST1 significantly decreased RABV CVS infection in SK-N-SH cells (P < .01, Figure 5C) with no cytotoxic effect (Supplementary Figure 4). These results indicate that _N_- and 6-_O_-sulfations of cellular HS are required for efficient RABV infection of target cells.

No Role of Heparan Sulfate in Infection With Rabies Virus High Egg Passage Flury Strain

We examined the interaction of HS to another strain of RABV, HEP, an attenuated virus strain [27]. The G of RABV HEP (HEP-G) shares 90.6% amino acid identity with CVS-G (GenBank accession numbers AB085828 and LC325820). Surprisingly, unlike CVS (Figure 1B and 1C), preincubation of HEP with either HS or heparin had no effect on viral infectivity in SK-N-SH cells (Supplementary Figure 5A and 5B). The HEP infection was also resistant to enzymatic degradation of cellular HS by treatment with heparinase III (Supplementary Figure 5C) in contrast with CVS infection (Figure 2C). These results indicate that the infection by RABV HEP strain of target cells is independent of cellular HS.

Identification of Critical Amino Acid Residues in Challenge Virus Standard G for the Interaction With Heparin

We examined the CVS-G region responsible for binding to HS using VSV pseudotyped with chimeric G mutants of CVS and HEP. VSVΔG* is a replication-incompetent VSV lacking the glycoprotein gene [24]. As reported [15], preincubation with heparin had no effect on the infectivity of VSVΔG* pseudotyped with VSV G (VSV-G) (Figure 6A). Furthermore, preincubation with heparin reduced the infectivity of VSVΔG* pseudotyped with CVS-G, but not HEP-G (Figure 6A), consistent with results of neutralizing assays with heparin using infectious RABV CVS and HEP (Figure 1C and Supplementary Figure 5B). We prepared a series of VSVΔG* pseudotyped with chimeric mutants between HEP-G and CVS-G, as shown in Figure 6B. Preincubation with heparin reduced the infectivity of VSVΔG* bearing HC(126–273), but not HC(1–125) and HC(274–407) (Figure 6C), indicating that the region between amino acid positions 126 and 273 of CVS-G was responsible for the recognition and neutralization by heparin. In addition, treatment with heparin neutralized the infectivity of VSVΔG* bearing HC(126–223) and HC(182–273), but not HC(126–181), HC(182–223), and HC(224–273) (Figure 6C), suggesting that multiple sites between CVS-G positions 126 and 273 are involved in the heparin neutralization.

Figure 6.

Effect of heparin treatment on the infectivity of pseudotyped replication-incompetent vesicular stomatitis virus (VSVΔG*). A, VSVΔG* pseudotyped with G proteins of vesicular stomatitis virus (VSV-G), high egg passage Flurry (HEP-G), or challenge virus standard (CVS-G) were pretreated with 100 μg/mL of heparin and then inoculated to SK-N-SH cells. Infection with pseudotyped VSVΔG* was identified by green fluorescent protein (GFP) reporter expression. Cellular nuclei were stained with Hoechst 33342. The numbers of GFP-positive cells and total cells were counted by imaging cytometry. Relative infectivity was calculated as a percentage of untreated conditions. B, Schematic representation of chimeras used in this study. The numbers indicate amino acid positions of mature G protein without the signal peptide. C and D, VSVΔG* pseudotyped with chimeric G (C) or point mutants of HEP-G (D) were pretreated with 100 μg/mL of heparin and then inoculated to SK-N-SH cells. Cells were analyzed by imaging cytometry, and relative infectivity was calculated as described above. The values in the graphs are shown as means ± standard deviations of triplicates from a representative experiment. Abbreviations: CVS-G, challenge virus standard G protein; HC, HEP/CVS chimeric glycoprotein; HEP-G, high egg passage Flurry G protein; TM, transmembrane domain; VSV-G, vesicular stomatitis virus G protein.

To determine residues of CVS-G targeted by heparin, we constructed a series of HEP-G mutants with multiple reciprocal substitutions between positions 126 and 273 and identified 3 candidates (Supplementary Figure 6). Preincubation with heparin decreased the infectivity of VSVΔG* pseudotyped with HEP-G mutants carrying multiple substitutions at positions 158, 186, and 248 [ie, HEP-G(158K/186R), HEP-G(158K/248K), HEP-G(186R/248K), and HEP-G(158K/186R/248K)], whereas VSVΔG* bearing HEP-G with single substitutions [ie, HEP-G(158K), HEP-G(186R), and HEP-G(248K)] were resistant to the treatment (Figure 6D). Conversely, we introduced multiple reciprocal substitutions at the same 3 positions of CVS-G and confirmed that 158N, 186G, and 248E substitutions confer additive resistance to preincubation with heparin to CVS-G–pseudotyped VSVΔG* (Supplementary Figure 7A). These results indicated that residues at positions 158, 186, and 248 define the differential susceptibility to preincubation with heparin in both CVS-G– and HEP-G–carrying viruses.

It has been reported that clusters of basic amino acids, such as lysine (K) or arginine (R), in viral proteins, mediate HS binding [18, 28, 29]. To examine the contribution of the basic residues 158, 186, and 248 of RABV G to heparin neutralization, we generated HEP-G mutants with basic or neutral amino acid substitutions at these positions. Preincubation with heparin reduced the infectivity of VSVΔG* pseudotyped with HEP-G mutants with basic amino acid substitutions (K or R), but not neutral amino acid substitutions (alanine [A]) at 186 and 248 (Supplementary Figure 7B). However, heparin neutralized VSVΔG* pseudotyped with HEP-G carrying either basic or neutral amino acid substitutions at 158 (Supplementary Figure 7C). These results indicate that the basic residues at 186 and 248 influence RABV neutralization by heparin, but residue 158 affects RABV infectivity by another mechanism(s).

Different Sensitivity of Rabies Virus G Proteins to Heparin Treatment

In addition to CVS and HEP, the sensitivity of RABV G to heparin treatment was examined using the G of Evelyn Rokitnicki Abelseth (ERA) and Nishigahara, fixed strains, and 1088, a street strain. Preincubation with heparin neutralized VSVΔG* pseudotyped with G of both Nishigahara and 1088, whereas the inhibitory effects were relatively weak compared with that of VSVΔG* bearing CVS-G (Supplementary Figure 8A). In contrast, preincubation with heparin had no effect on the infectivity of VSVΔG* bearing G of ERA (Supplementary Figure 8A). Multiple sequence alignment based on glycoproteins of RABV fixed strains (CVS, HEP, ERA, Nishigahara) and street strains (1088, 9147FRA, 8764THA) showed that K158 is conserved but R186 and K248 are not conserved between the glycoproteins of RABVs sensitive to heparin pretreatment (ie, CVS, Nishigahara, and 1088) (Supplementary Figure 8B). These results suggest that G of some RABV strains recognizes heparin but the molecular binding modes are different from that of CVS.

DISCUSSION

Previous studies implicated the presence of RABV attachment factors in the cellular membrane fraction; however, no specific molecule has been identified [2]. Here, we show that cellular HS supports RABV CVS infection (Figure 2). We also have identified CVS-G as a ligand for HS and characterized the interaction between HS and CVS-G (Figure 4). Based on these results, we conclude that cellular HS functions as the first identified attachment host factor for RABV infection through interaction with the viral glycoprotein. A previous study showed that the exogenous expression of RABV receptor confers susceptibility to RABV infection of nonpermissive cells [4]. The ubiquitous expression of HS on cells permissive and nonpermissive for RABV indicates that HS serves an attachment factor rather than entry receptor for RABV infection.

The interactions with HS and proteins are partially mediated by an electrostatic binding between negatively charged chains of HS and basic amino acids in the target proteins. In addition, some viral proteins require specific _N_- or _O_- sulfation for HS interaction [17, 30–34]. Indeed, we have demonstrated that RABV CVS requires _N_- and 6_O_-sulfations of HS, but not 2_O_-sulfation, for the interaction with HS and efficient infection of cells (Figure 5). These observations strongly support that RABV CVS certainly recognizes cellular HS and uses it for infection.

The antiviral effects of heparin on RABV infection have been considered to be related to competition for the binding to NCAM, a RABV receptor [4]. Our study proposes an alternative mechanism whereby heparin inhibits RABV cell surface attachment through the direct binding to RABV (Figure 3B). Notably, the neutralization effect of preincubation of heparin with RABV CVS was much stronger than that of preincubation of heparin with cells, and the cell surface attachment of RABV CVS was decreased in the presence of heparin (Figure 1C and 1E). These results indicate that the anti-RABV effect of heparin mainly depends on the attachment inhibition of RABV on the cellular surface through its interaction with RABV. We also demonstrated that Huh-7 and A549 cells are deficient in NCAM expression but susceptible to CVS infection (Figure 1 and Supplementary Figure 1), indicating that the expression of NCAM is unnecessary for RABV infection of these cells. Importantly, the NCAM-independent antiviral effect of heparin was also clearly observed in these NCAM-negative cells (Figure 1G and Supplementary Figure 1C).

Reciprocal amino acid substitutions at positions 158, 186, and 248 switched the different susceptibility to heparin treatment between CVS-G and HEP-G (Figure 6D and Supplementary Figure 7A), suggesting these residues are responsible for the recognition by cellular HS. Other studies revealed that some viruses possess basic residues critical for the interaction with HS in their structural proteins [18, 28, 29]. Site-directed mutagenesis of RABV G demonstrated that the basic residues at 186 and 248 are key for the interaction of G with HS (Supplementary Figure 7B). Conversely, residue 158 seems to be involved in the HS interaction by a different mechanism(s) (Supplementary Figure 7C). The asparagine (N) residue at 158 of HEP-G forms the consensus motif for N-linked glycosylation (N-x-S), which promotes RABV production [35]. The reciprocal substitution (158N) in CVS-G introduced a potential N-linked glycosylation site at the region (ie, K-C-S to N-C-S). Therefore, a possible explanation is that N-linked glycosylation at position 158 interferes with the interaction of G to HS.

Heparin neutralized VSVΔG* pseudotyped with G of both fixed and street strains of RABV except for HEP and ERA (Figure 6 and Supplementary Figure 8A). Indeed, RABV HEP was resistant to preincubation with soluble HS and heparin and infected cells independently of cellular HS (Supplementary Figure 5). These results suggest that certain RABV strains use cellular HS as an attachment factor; however, others, such as HEP and ERA, are likely to use other unknown molecules for cellular attachment. We largely used the fixed strains of RABV for this study. Further studies are needed to examine the interaction of HS with other strains, especially circulating street strains of RABV.

Previous studies demonstrated that the ability to bind HS influences the pathogenicity of several viruses [12, 14, 36–41]. Field isolates of eastern equine encephalitis virus bind to HS, and this interaction contributes to neurovirulence [14]. In addition, a single mutation in E2 protein confers HS-binding property and increased neurovirulence of Sindbis virus [36]. In contrast, HS binding is often involved in virulence attenuation of cell culture–adapted viruses [12, 37–41]. Notably, our heparin neutralization assays suggest that pathogenic strains of RABV (ie, CVS, Nishigahara, 1088), but not attenuated strains (HEP, ERA), bind to HS (Supplementary Figure 8A). The contribution of HS-binding property to the pathogenicity of RABV requires further studies.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank Dr Chang-Kweng Lim, Department of Virology I, National Institute of Infectious Diseases, Japan, for providing the rabies virus HEP strain, and Dr Akira Nishizono, Department of Microbiology, Oita University, Japan, for providing pCI-1088-G, a rabies virus 1088 glycoprotein expressing vector. We also thank RIKEN BioResource Center, Tsukuba, Japan, for providing SK-N-SH, A549 and Huh-7 cells.

Financial support. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (15K07716); the Japan Agency for Medical Research and Development (AMED)/Japan International Cooperation Agency (JICA) within the framework of the Science and Technology Research Partnership for Sustainable Development (SATREPS); and by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H06429, 16H06431, 16K21723).

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Presented in part: 160th Meeting of the Japanese Society of Veterinary Science, Kagoshima, Japan, September 2017. Abstract DVO-95; 65th Annual Meeting of the Japanese Society for Virology, Osaka, Japan, October 2017. Abstract P2-N1-04.

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Author notes

Present address: Toyoyuki Ose, Laboratory of X-Ray Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.

© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about\_us/legal/notices)

Topic:

• heparin
• glycoproteins
• heparitin sulfate
• rabies
• rabies virus
• infections
• viruses
• pathogenicity

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