Timing of galectin-1 exposure differentially modulates Nipah virus

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Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytia formation in 1

endothelial cells 2

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Running Title: Nipah virus infection regulated by galectin-1 4

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Omai B. Garner1, Tatyana Yun2, Olivier Pernet3, Hector C. Aguilar4, Arnold Park3, Thomas A. 6

Bowden5, Alexander N. Freiberg2, Benhur Lee3,6*, Linda G. Baum1* 7

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1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los 9

Angeles, CA, USA 10

2Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA 11

3Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of 12

Medicine at UCLA, Los Angeles, CA, USA 13

4Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State 14

University, Pullman, WA, USA 15

5Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, 16

Roosevelt Drive, Oxford OX3 7BN, United Kingdom 17

6Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 18

*These authors contributed equally 19

Corresponding authors: [email protected] and [email protected] 20

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JVI Accepts, published online ahead of print on 10 December 2014J. Virol. doi:10.1128/JVI.02435-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 22

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human 23

endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have 24

previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F 25

mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and 26

hemorrhage. Galectin-1 is an endogenous carbohydrate binding protein that binds to specific glycans 27

on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of 28

NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for 29

example, galectins can promote attachment of HIV to T cells and macrophages and attachment of 30

HSV-1 to keratinocytes, but can also inhibit influenza entry into airway epithelial cells. Using live 31

Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and 32

infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell 33

glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial 34

phase of virus attachment; in contrast, addition of galectin-1 post-infection results in reduced 35

production of progeny virus and syncytia formation. Thus, galectin-1 can have dual and opposing 36

effects on NiV infection of human endothelial cells. While various roles for galectin family members 37

in microbial-host interactions have been described, we report opposing effects of the same galectin 38

family member on a specific virus, with the timing of exposure during the viral life cycle determining 39

the outcome. 40

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Importance 42

Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high 43

mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting 44


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catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well 45

understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition 46

receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by 47

microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed 48

endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to 49

the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, 50

depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects 51

of a host lectin for a virus in one type of host cell. 52

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Introduction 57

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that targets endothelial and neural 58

cells. Infection with NiV can result in a severe encephalitic or respiratory syndrome with case fatality 59

rates ranging from 40-100% in humans [1, 2]. While initial outbreaks involved transmission from bats-60

to-pigs, and then from pigs-to-humans, more recent outbreaks have been shown to involve human-to-61

human virus transmission [2]. There are currently no approved vaccines or anti-viral agents targeting 62

NiV for human cases, and quarantine has been the predominant measure to restrain the spread of the 63

virus[3]. 64

NiV preferentially infects microvasculature endothelial cells that express the entry receptor 65

ephrinB2 [4]. The NiV attachment protein NiV-G binds to ephrinB2 or ephrinB3 [5]on the host cell 66

plasma membrane, triggering the NiV fusion protein, NiV-F, to execute fusion of the viral envelope 67

with the host cell membrane. Infected microvascular endothelial cells produce NiV-F and NiV-G 68

which results in fusion of the cells into syncytia[6]; this syncytia formation contributes to vascular 69

compromise and hemorrhage, two hallmarks of Nipah virus infection [7]. 70

Endothelial cells respond to viral infection by producing inflammatory mediators including 71

cytokines, which regulate leukocyte trafficking into adjacent tissues, and by presenting viral antigens 72

[8]. Among the inflammatory mediators produced by endothelial cells are the galectins, a family of 73

mammalian lectins implicated in immune regulation. Vascular endothelial cells express several 74

galectin family members, including galectins-1 and -9. For example, infection of vascular endothelial 75

cells with Ebola virus results in endothelial cell activation [9] and increased expression of galectin-1 76

[10, 11]. Infection of endothelial cells with Dengue virus induces expression of galectin-9 [12], as does 77

binding of double stranded RNA to TLR-3 on endothelial cells [13]. 78


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Galectins play several roles in modulating host-pathogen interactions. Galectins can participate 79

in innate immune recognition and clearance of pathogens, however pathogens can also use galectins as 80

attachment receptors. Galectins specifically recognize a subset of microbes based on binding to 81

pathogen specific polysaccharides [14]. For example, galectin-4 and galectin-8 bind to and directly kill 82

human blood-group B expressing Escherichia coli [15] and galectin-3 binds to and directly kills 83

Candida albicans [16] in the absence of immunoglobulin or complement. In contrast, Pseudomonas 84

aeruginosa and Trypanosoma cruzi use galectin-3 to bind to and infect human cells, and galectin-1 on 85

human cervical epithelial cells is an attachment factor for Trichomonas vaginalis [17-19]. Similarly, 86

galectin-1, which preferentially recognizes LacNAc (Gal1,4GlcNAc) containing glycans, increases 87

human immunodeficiency virus (HIV) infection of monocyte-derived macrophages through 88

stabilization of virus attachment [20-22] and galectin-9 promotes HIV infection of T cells by 89

regulating the cell surface redox status [23]. Thus, different galectins can have pleiotropic and 90

sometimes opposing roles in the etiology and pathophysiology of microbial infections. However, 91

opposing effects of a single galectin on a specific virus have not been reported. 92

Using NiV-F/G pseudotyped particles, our labs previously demonstrated that galectin-1 93

produced by endothelial cells can inhibit endothelial cell syncytia formation mediated by NiV-G and 94

NiV-F [11, 24]. In these studies, galectin-1 reduced NiV-F mediated fusion of endothelial cells by 95

interfering with NiV-F maturation, reducing lateral mobility of NiV-F on the plasma membrane, and 96

inhibiting the conformational change in NiV-F triggered by receptor binding to NiV-G that is 97

necessary for cell-cell fusion [11]. While galectin-1 produced by endothelial cells can clearly inhibit 98

post-infection cell-cell fusion triggered by NiV-F, our prior studies did not address the effect of 99

galectin-1 on NiV attachment and entry into host cells. In the present study, we found that galectin-1 100

can enhance live NiV attachment to and infection of endothelial cells by bridging glycans on the viral 101


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envelope to cell surface glycoproteins. While the presence of galectin-1 enhances NiV entry, galectin-1 102

also inhibits syncytia formation of infected cells. In both cases, a specific N-linked glycan site on NiV-103

F plays a critical role in mediating galectin-1’s effects. Our results highlight the complex roles 104

galectin-1 may play in viral pathogenesis. 105

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Materials and Methods 107

Cells, virus, and reagents 108

Vero cells (ATCC) were maintained in MEM alpha (Invitrogen) with 10% FBS (Hyclone) and 109

2mM Glutamax in 5%CO2 at 37C. 293T cells were maintained in DMEM (Invitrogen) with 10% FBS 110

(Hyclone) and 2mM Glutamax. VeroCCL-81 cells were maintained in DMEM (Gibco) with 10% FBS 111

(Hyclone). HUVECs (BD Biosciences) were maintained in MDCB-131 complete media with fetal 112

bovine serum and antibiotics (VEC Technologies, INC). HUVECs (LONZA) were maintained in 113

Endothelial Basal Medium-2 (EGM-2) with EGM-2 SingleQuots. 114

Recombinant human galectin-1 was expressed in E. coli and purified by affinity 115

chromatography on lactosyl-Sepharose, as described [25]. Galectin-1 has unpaired thiols in the 116

carbohydrate recognition domain that can form disulfide bonds and inactivate the lectin, so galectin-1 117

is prepared and stored in PBS with dithiothreitol (DTT) and, in all assays, buffer controls are the stock 118

PBS with 1.2mM DTT at the appropriate dilution [26]. 119

NiV-F and G were pseudotyped onto a reporter vesicular stomatitis virus (VSV) expressing 120

Renilla luciferase as in [27]. Nipah virus (NiV) strain Malaysia (NiV-MAL) was kindly provided by the 121

Special Pathogens Branch, CDC, Atlanta, GA, and propagated in VeroE6 cells. All work with 122

infectious NiV was carried out under Biosafety Level 4 (BSL-4) conditions in the Robert E. Shope 123

BSL-4 and in the Galveston National Laboratory BSL-4 laboratories at the University of Texas 124

Medical Branch (UTMB). 125

126

Quantitation of viral entry 127

Cells plated in 48-well plates were infected with pseudotyped virions in PBS plus 1% FBS for 128

2 hours (hrs) at 25oC at 2000rpm (spinoculation). After 2 hr, cells were washed, appropriate growth 129


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medium was added, and cells were incubated at 37oC. At 24 hr post-infection, cells were lysed and 130

luciferase activity was measured as relative light units (RLU) using a Renilla luciferase detection 131

system (Promega, Madison, WI) and a Veritas microplate luminometer (Turner Biosystems, 132

Sunnyvale, CA). Galectin-1, buffer control, 100mM lactose and 100mM sucrose were added just prior 133

to spinoculation. 134

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Reduction in complex N-glycans 136

Cells (Vero, 293T, HUVECs) were treated with kifunensine (2g/ml) for 24 hrs. To confirm 137

loss of complex N-glycans on the cell surface, cells were suspended in PBS with 5mM 138

ethylenediaminetetraacetic acid (EDTA), washed, and resuspended in biotinlyated L-PHA in PBS 139

(1:1000) plus 1% bovine serum albumin (BSA) (Gemini Scientific) for 1 hr at 4oC. Cells were washed 140

in PBS and bound PHA detected with a FITC-conjugated streptavidin (SA) (1:60) in PBS plus 1% 141

BSA, for 1 hr at 4C. Cells were washed in PBS with 1% BSA and flow cytometric analysis was 142

performed on a FACScan, using CellQuest software (Becton-Dickenson). Pseudotyped VSV 143

expressing NiV-G and NiV-F were produced by infecting kifunensinee-treated or control cells with 144

VSV-rLuc virions for 24 hr. Supernatents were collected and virions purified by centrifugation through 145

20% sucrose in NTE buffer. To examine loss of N-glycan processing by immunoblot, NiV-G and 146

NiV-F were separated and detected as previously described [24]. 147

148

Rescue of recombinant Nipah virus (rNiV) and in vitro luciferase assay 149

Recombinant Nipah virus expressing secreted Gaussia luciferase (GLuc) (rNiV-GLuc) was rescued at 150

BSL-4 [28]. Briefly, HEK 293T cells were transfected with the following plasmids: T7-driven 151

positive-sense rNiV antigenome (3.5g), codon-optomized pCAGGS/T7 (1g), and the T7-driven 152


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support plasmids encoding NiV nucleoprotein (1g), phosphoprotein (0.2g) and polymerase (0.4g). 153

Transfection was performed using TransIT-LT1 transfection reagent (Mirus) following the 154

manufacturer’s instructions. After 4 days post-transfection, Gaussia luciferase expression was 155

confirmed using cell supernatant and the BioLux Gaussia Luciferase assay kit (New England Biolabs). 156

Collected supernatant was used for subsequent virus preparation in Vero CCL-81 cells. HUVECs were 157

infected with rNiV-GLuc at different multiplicity of infections (MOIs) and cells supernatants were 158

collected at the indicated intervals. Luciferase assay were performed using BioLux Gaussia Luciferase 159

assay kit (New England BioLabs) according to the manufacturer’s protocol and expression was 160

measured on Modulus Luminometer (Turner BioSystems, Inc). 161

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Nipah virus plaque assay 163

For titrations, confluent monolayers of VeroCCL-81 cells were infected with 100μl of serial 164

tenfold dilutions of virus-containing cell supernatant. After 1 h incubation at 37°C and 5% CO2, the 165

inocula were removed and wells overlaid with a mixture of one part 1.0% methylcellulose (Fisher 166

Scientific) and one part 2xMEM (Gibco, Invitrogen) supplemented with 2% FBS and 2% 167

penicillin/streptomycin. The plates were incubated at 37°C and 5% CO2 for 3 days and stained with 168

0.25% crystal violet in 10% buffered formalin. Plates were washed and plaques enumerated. 169

170

Wild-type Nipah virus, galectin-1 experiments 171

HUVECs were infected with NiVMAL at a MOI of 0.1 for 1hr at 37oC. Infections were 172

performed either in the presence of various galectin-1 concentrations (1, 7, and 20µM) or cells were 173

treated with galectin-1 post-infection. In the case of infections performed in the presence of galectin-1, 174

the inoculum was removed 1 hpi and cells were washed three times with 0.1M β-Lactose, followed by 175


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incubation in complete EGM-2 media. Cells infected only with NiV were washed three times with 176

media and incubated in complete EGM-2 media containing different concentrations of galectin-1 (1, 7 177

or 20µM). Supernatant aliquots were harvested at 1, 12, 24 and 36 hpi and stored at -800C until 178

titration. DTT was used as negative control and experiments were performed in triplicates. 179

For DAPI staining, HUVECs were grown on 12-well plate and incubated with NiV for 1 h at 180

37°C. Galectin-1 treatment was performed as described above. Twenty-four hpi, cells were fixed in 181

10% buffered formalin, nuclei stained with DAPI (Sigma) 20µg/ml and analyzed by fluorescence 182

microscopy. 183

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Results 187

188

Galectin-1 increases viral infection of endothelial cells 189

Galectin-1 inhibits endothelial cell syncytia formation mediated by NiV envelope 190

glycoproteins[11]. Although galectin-1 can bind to both NiV-F and –G, we showed that inhibition of 191

cell-cell fusion was mediated largely by galectin-1 binding to abundant and accessible lactosamine 192

moieties on complex N-glycans at the “F3” N-glycan site (93NNT101) on NiV-F[11] [24]. The F3 N-193

glycan site is close to the cathepsin L cleavage site 103DLVGDVR109. Binding of dimeric galectin-1 to 194

F3 N-glycans on different NiV-F protomers likely accounts for the suite of inhibitory activities that 195

galectin-1 exerts on NiV-F mediated fusion[11]. We therefore expected galectin-1 to inhibit NiV 196

infection just as potently. Surprisingly, when we examined the function of galectin-1in Nipah virus 197

entry using NiV-F/G pseudotyped particles (NiVpp) [29], we found that addition of recombinant 198

galectin-1 significantly increased infection of target cells in a concentration dependent manner (Fig. 199

1A). 200

We confirmed that the galectin-1 mediated increase in infection was carbohydrate dependent. 201

Lactose, but not sucrose, specifically abrogated the galectin-1 mediated increase in infection, while 202

lactose alone in the absence of galectin-1 had no effect (Fig. 1B). Thus, exogenous galectin-1 increased 203

viral infection in a dose-dependent and carbohydrate binding-dependent manner. 204

205

Galectin-1 increases viral attachment to host cells 206

Entry of NiV into host cells requires two steps, viral attachment to the target cell surface 207

mediated by NiV-G followed by membrane fusion mediated by NiV-F. Viral attachment is largely 208

energy-independent while virus-cell fusion is an energy-dependent process [30]. To examine the point 209


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at which galectin-1 enhancement occurs, NiVpp was centrifuged onto target cells in the presence or 210

absence of galectin-1 for 2 hours at 4oC, to allow viral attachment but not virus-target cell fusion. 211

Alternatively, virus was centrifuged onto target cells in the absence of galectin-1, and then incubated 212

with or without galectin-1 for 1 hr at 37oC to permit fusion. As shown in Fig. 1C, galectin-1 present 213

during the 4oC spinoculation step clearly enhanced viral entry. In contrast, galectin-1 added after 214

spinoculation at 4oC and present only during the 37oC fusion step, did not increase viral entry. These 215

experiments suggest that galectin-1 increased infection by enhancing viral attachment to host cells. 216

Moreover, that no inhibition was seen suggests that there are functional NiV-F envelope spikes on the 217

virion particles that are not bound by galectin-1 and are still capable of being triggered by receptor 218

binding to NiV-G. 219

220

Galectin-1 bridges N-glycans on viral envelope glycoproteins to glycoproteins on target cells 221

Galectin-1 stabilizes HIV-1 attachment and adsorption to host cells by binding specifically to 222

clustered complex N-glycans on gp120 and CD4, bridging the virus to the target cell [20-22]. We 223

asked if complex N-glycans, that bear glycan ligands recognized by galectin-1, were involved in the 224

galectin-1 mediated increase in viral entry of NiVpp. To eliminate complex N-glycans from viral 225

envelope glycoproteins, virus was produced in 293T cells treated with kifunensine, to block processing 226

of high-mannose N-glycan precursors into complex N-glycans [31]. We detected equivalent expression 227

levels of NiV-F by immunoblot from cells treated with and without kifunensine (data not shown). We 228

confirmed that kifunensine blocked N-glycan processing in 293T cells by flow cytometry with the 229

lectin L-PHA (Fig. 2A); loss of L-PHA binding indicates loss of complex N-glycans. 230

Vero cells were infected with NiVpp virus, with NiVpp virus lacking complex N-glycans from 231

kifunensine-treated 293T cells, or with NiVpp virus specifically lacking the NiV-F3 N-glycan in the 232


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presence or absence of exogenous galectin-1 (Fig. 2B). As expected, addition of galectin-1 resulted in 233

a 12.6 fold increase in target cell infection of virus with a full complement of complex N-glycans (Fig. 234

2B “wt NiVpp”. Remarkably, the loss of the F3 N-glycan site on NiV-F resulted in a >80% reduction 235

in galectin-1 mediated enhancement (3.2 fold vs 12.6 fold enhancement for NiV-F3pp vs wt NiVpp 236

respectively), underscoring the contribution of the complex N-glycan at the F3 site in the enhancement 237

of virus entry as well as fusogenicity [11, 27]. However, the galectin-1 mediated increase was further 238

reduced (~95% reduction) when cells were infected with virus lacking complex N-glycans (Fig. 2B, 239

“Complex N-glycanless NiVpp”), indicating that other complex N-glycans on NiV-F or NiV-G also 240

contributed to the galectin-1 enhancement effect. 241

To examine the role of host cell complex N-glycans in galectin-1 mediated enhancement of 242

viral infection, we treated uninfected Vero cells with kifunensine to reduce complex N-glycans on the 243

target cells. A reduction of complex N-glycans was confirmed by flow cytometry with L-PHA, as 244

described above (Fig. 2C). A greater than ten-fold reduction in complex N-glycans from target Vero 245

cells reduced galectin-1 mediated enhancement of infection by approximately 30% (Fig. 2D). Thus, 246

complex N-glycans on host cell glycoproteins participate in the galectin-1 enhancement of viral 247

infection; however, as ten-fold reduction of complex N-glycans on kifunensine treated host cells 248

partially reduced the galectin-1 enhancement effect, other host cell glycans, likely O-glycans, may also 249

play a role on the host cell side [32]. Collectively, the data in Fig. 2 demonstrate that glycans on both 250

the viral envelope and on the target cell surface are important for galectin-1 enhancement. Our results 251

support a model in which the enhancement effect results from dimeric galectin-1 acting as a bridge or 252

bivalent attachment factor between the virus and the target cell, with complex N-glycans contributing 253

to attachment on both the virus and the host cell. 254

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Galectin-1 enhances Nipah virus infection of human endothelial cells 256

Endothelial cells are primary targets of NiV infection in vivo, due to expression of the viral 257

attachment receptor ephrinB2 [4]. To determine if galectin-1 would enhance Nipah virus infection of 258

physiologically relevant target cells, we infected primary human umbilical vein endothelial cells 259

(HUVECs) with NiVpp (Fig. 3A) in the presence or absence of exogenous galectin-1. The addition of 260

galectin-1 resulted in a 6-fold increase in infection of HUVECs, similar to the effect we had observed 261

with Vero cells as targets of infection. 262

Thus far, we used NiVpp, rather than replication competent, wild-type infectious NiV. NiVpp 263

is a VSV-based pseudotyped virus capable only of single cycle of infection, and the bullet-shaped 264

VSV-based particles may not fully recapitulate the morphology or envelope glycoprotein densities 265

present on native pleomorphic paramyxovirus particles [33]. To determine if galectin-1 modulates 266

infection by replication competent NiV, we first generated and rescued, under BSL4 conditions, a 267

recombinant NiV expressing secreted Gaussia luciferase (rNiV-GLuc) (see Materials and Methods). 268

To facilitate the generation of additional recombinant NiVs such as the one bearing the F3 N-glycan, 269

we also developed an efficient and robust reverse genetics system for henipaviruses [28]. 270

271

This rNiV-GLuc replicated to the same end-point titers as the parental NiV Malaysia strain 272

(data not shown) and was pathogenic in a lethal hamster challenge model [34]; using rNiV-GLuc 273

allowed us to accurately quantify enhancement or inhibition of live virus entry by sampling the 274

infected cell culture supernatant for GLuc activity. We first asked if rNiV-GLuc would also exhibit 275

enhanced infection of endothelial cells in the presence of galectin-1. HUVEC cells infected at an MOI 276

of 0.05 with rNiV-GLuc showed a dose-dependent enhancement of infection in the presence of 277

galectin-1 at 24 hours post infection (hpi). The enhancement effect plateaued at 7-10 M galectin-1, 278


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with a 6-fold increase in infection compared to controls lacking exogenous galectin-1 (Fig. 3B). This 279

level of enhancement was similar to that observed for NiVpp in HUVECs, albeit in the presence of 280

20M galectin-1. 281

Next we reasoned that, if galectin-1 was bridging virions to the cell surface, as suggested by the 282

data in Fig. 2, then the relative level of enhancement should be increased when the amount of viral 283

inoculum was decreased. That is, when the number of infectious viral particles is more limiting, the 284

effect of galectin-1 on bridging viruses to the cell surface should become more apparent. Thus, we 285

reduced the viral inoculum by 5-fold and quantified the corresponding effect of galectin-1 on virus 286

infection. Indeed, at a MOI of 0.01, 10M of galectin-1 enhanced rNiV-GLuc infection by up to 40-287

fold (Fig. 3C, black bars) compared to the 6-fold enhancement seen with an equivalent dose of 288

galectin-1 when a higher viral inoculum (MOI of 0.05) was used (Fig 3B). 289

290

Galectin-1 enhances rNiV-GLuc virus infection by bridging complex N-Glycans 291

To confirm that galectin-1 enhances rNiV-GLuc infection by bridging complex N-glycans 292

present on virions and the endothelial cell surface, as we had seen in infection of Vero cells with 293

NiVpp (Fig. 2), we generated either HUVECs or viruses deficient in complex N-glycans by treating 294

HUVECs with kifunensin (Cells Kif+) or growing rNiV-GLuc in the presence of kifunensin (Virus 295

Kif+). Complex N-glycanless Nipah virus (Virus Kif+) was equally infectious when tittered on Vero 296

cells (data not shown). As shown in Fig. 3C, depletion of complex N-glycans on target cells (Cells 297

Kif+) moderately reduced galectin-1 enhanced rNiV-GLuc infection of HUVECs (black bars versus 298

dark grey bars), as we had seen with Vero cells. Depleting virus of complex N-glycans (Virus Kif+) 299

also reduced galectin-1 enhancement of infection (light grey bars) more significantly, as we had seen 300

with NiVpp. When both virus and HUVECs lack complex N-glycans (Virus Kif+/Cells Kif+), 301


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galectin-1 had the least enhancement effect (white bars). In toto, our data reveal that galectin-1 302

enhanced infection of both rNiV-GLuc and NiVpp by the same mechanism, i.e. the interaction 303

between galectin-1 and Nipah virus results primarily from galectin-1 binding to complex N-glycans on 304

NiV envelope glycoproteins, and that enhancement of infection involves galectin-1-mediated bridging 305

of viral glycans to glycans on the host cell surface. The remaining degree of galectin-1 mediated 306

enhancement seen with kifunensine treated cells and virus suggest that lactosamine residues on O-307

linked glycans might also serve as bridging receptors for galectin-1’s infection enhancement effect. 308

However, the role of these putative O-glycans may only be unmasked when cognate complex N-309

glycans are limiting. 310

311

Timing of galectin-1 exposure has opposing effects in Nipah virus replication 312

Previous work in our labs has shown that galectin-1 inhibited syncytia formation mediated by 313

NiV-F+G expressed by endothelial cells [11, 24]. Galectin-1 reduced NiV-F mediated fusion of 314

endothelial cells by retarding NiV-F maturation, reducing the lateral mobility of NiV-F in the plasma 315

membrane, and inhibiting triggering of NiV-F that results in fusion of the virus with the host cell 316

plasma membrane. Moreover, endogenous galectin-1 produced by endothelial cells was sufficient to 317

inhibit syncytia formation [11]. Importantly, all of the galectin-1 mediated effects that we had 318

previously analyzed would occur following Nipah virus infection, and was studied only in a cell-cell 319

fusion context. Thus, while the data presented demonstrate that galectin-1 enhances viral entry when 320

present at initial infection, we asked if downstream events, i.e. syncytia formation and viral replication 321

in human endothelial cells infected with NiVMAL, were affected by the presence of galectin-1. 322

To examine the effect on viral replication, HUVECs infected with the Nipah virus strain 323

Malaysia (NiVMAL) were incubated post-infection with galectin-1 or buffer control. The presence of 324


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exogenous galectin-1 post-infection at early time points (12 and 24 hpi) inhibited virus replication 325

titers below the limit of detection, effectively reducing viral titers by >3 logs at 24 hpi. This inhibitory 326

effect of galectin-1 persisted at 36 hpi as a 15-fold reduction in viral titers (Fig. 4A). 327

To examine the effect of galectin-1 on syncytia formation, wild type NiVMAL-infected 328

HUVECs were analyzed microscopically in three conditions; no exogenous galectin-1 added, galectin-329

1 added before infection, or galectin-1 added post-infection. Addition of exogenous galectin-1 before 330

infection enhanced syncytia formation (Fig. 4B, panel 2), consistent with the effect of galectin-1 on 331

increasing NiV infection of endothelial cells (Fig. 3). In contrast, addition of exogenous galectin-1 332

after NiV infection essentially abrogated syncytia formation (Fig. 4B, panel 3). This inhibitory effect 333

of galectin-1 on endothelial cell fusion (quantified in Fig. 4C) is consistent with our previous 334

observation using pseudotyped virus [11]. 335

To determine if the NiV-F3 N-glycan contributes to the differential effect of galectin-1 on 336

syncytia formation in the context of live virus infection, we generated and rescued a rNiV-GLuc 337

bearing the NiV-F3 N-glycan mutant. Infection with the rNiV-GLuc F3 N-glycan mutant virus resulted 338

in an overall increase in syncytia formation, compared to wild-type NiV-MAL, consistent with the 339

hyperfusogenic phenotype previously observed for the NiV-F3 mutant [27]. However, addition of 340

galectin-1 before infection no longer enhanced syncytia formation, in contrast to the enhancement seen 341

with the wild-type virus in the presence of galectin-1 (Fig. 4C). Galectin-1 was still able to inhibit 342

syncytia formation by the mutant virus when added after infection, although lack of N-glycans at the 343

F3 site made the mutant virus more resistant to the inhibitory effect of galectin-1 (Fig. 4C). 344

Collectively, our data indicate that the timing of galectin-1 exposure differentially modulates 345

Nipah virus entry, syncytia formation and replicative spread in endothelial cells. Galectin-1 present at 346

the attachment/entry step of viral infection enhanced NiV entry into endothelial cells and also 347


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enhanced syncytia formation, likely due to increased virus envelope production in initially infected 348

cells. In contrast, galectin-1 added after NiV infection of endothelial cells inhibited both syncytia 349

formation and the replicative spread of progeny virus such that viral titers at 24hpi and 36hpi lagged by 350

at least 4 logs and 1 log respectively. 351

352


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Discussion 353

Innate immune mediators such as galectins have many functions, including triggering 354

endothelial activation, inducing cytokine production and release, promoting leukocyte infiltration of 355

infected tissues, and mediating pathogen clearance [10, 14, 16, 35, 36]. As galectin-1 can recognize 356

both self (mammalian) and non-self (pathogen) glycans, depending on factors such as glycan ligand 357

density and presentation [37], galectin-1 has been proposed to be both a damage-associated molecular 358

pattern (DAMP) that is released from injured cells, and a pattern recognition receptor (PRR), 359

recognizing pathogen associated molecular patterns (PAMPs), such as specific viral glycans [11]. 360

Thus, galectin-1 can have dual functions in innate immunity [38]. Our current data also demonstrate 361

that galectin-1 has complex and dichotomous roles in NiV infection. Galectin-1 enhanced initial NiV 362

attachment to and entry into human endothelial target cells (Fig. 3), while subsequently inhibiting cell 363

syncytia formation (Fig. 4) [11, 24], a key sequel of viral replicative spread. The ability of galectin-1 to 364

produce these divergent effects depends on when it is present during the viral replicative life cycle. 365

There are several examples of galectin-1 having complex or opposing roles in different viral 366

infections, via binding to glycan ligands on both the virus and the target cell. Galectin-1 bound to 367

influenza virus reduced virus entry into host cells; moreover, intranasal administration of galectin-1 368

enhanced survival of mice infected with a lethal dose of influenza virus, and galectin-1 null mice were 369

more susceptible to influenza virus infection than wildtype mice [39]. In contrast, galectin-1 increased 370

HIV-1 infection of monocyte derived macrophages through stabilization of virus attachment and 371

adsorption [20, 21]. That galectin-1 stabilized HIV adsorption to macrophages suggests that dimeric 372

galectin-1 can act as a bridge between virus glycans and host cell surface glycans, although this was 373

not directly demonstrated in those studies. 374


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Here we examined whether galectin-1 formed a bridge between viral particles and host cells to 375

enhance infection. The enhancement effect was clearly glycan-dependent, as the specific inhibitor 376

lactose, but not the non-cognate disaccharide sucrose, blocked the enhancement of viral infection (Fig. 377

1B). To specifically address the step at which galectin-1 binding promoted infection, we bound virus to 378

host cells at 4oC and initiated viral entry at 37oC (Fig. 1C). Galectin-1 added only during the 379

attachment step at 4oC enhanced infection, while galectin-1 added after viral attachment, but during 380

fusion at 37oC, did not enhance infection. These data support a model whereby galectin-1 enhancement 381

occurs as a result of bridging glycoproteins on the virus to glycoproteins on the host cell. To determine 382

whether the essential glycans recognized by galectin-1 were on the virus, the host cell, or both, we 383

eliminated complex N-glycans on viral particles and on the surface of the target cells by treatment with 384

the -mannosidase I inhibitor kifunensine. Complex N-glycans on viral glycoproteins were clearly 385

required for galectin-1 enhancement of infection (Fig. 2B and 3C). We have previously demonstrated 386

that galectin-1 specifically interacts with the F3 N-glycan on the NiV-F glycoprotein, and that the F3 387

N-glycan site is occupied by complex N-glycans that bears cognate ligands for galectin-1 [11, 27]. In 388

the present report, we show that this F3 glycan was the most significant viral determinant of galectin-1 389

mediated enhancement of infection (Fig. 2B and Fig. 4C). On the target cells, both complex N-glycans 390

and O-glycans appear to contribute to galectin-1 enhancement of infection (Fig. 2D and 3C) [40]. 391

While several endothelial cell surface glycoprotein receptors for galectin-1 have been identified [41], 392

the entire repertoire of cell surface glycoproteins that could bind to galectin-1 to facilitate viral 393

“bridging” and increase virus attachment is not known. In addition, little is known about the N- or O-394

glycan composition of the Nipah cellular receptor ephrinB2 [42] and whether galectin-1 can bind to 395

ephrinB2 on the surface of endothelial cells. 396


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Lectin-glycan interactions play complex roles in virus-host pathogenesis, and other types of 397

innate immune lectins have apparently dichotomous roles in viral pathogenesis. For example, mannan-398

binding lectin (MBL), a soluble innate immune lectin, binds to influenza A virus and leads directly to 399

virus inactivation [43]. In contrast, another mannose-binding human lectin, macrophage mannose 400

receptor (MMR) potentiates HIV binding to target cells [44-46], and MMR can also potentiate 401

infection of macrophages by influenza virus [47], so that different mannose-binding lectins can bind to 402

viruses to either reduce or potentiate infection. Considering the long-standing evolutionary relationship 403

between virus and host, it is not entirely surprising that viruses have learned to co-opt host innate 404

immune defenses for increased target cell entry and replication. 405

In the present study, we demonstrate that galectin-1 enhanced infection of endothelial cells by 406

live NiV (Fig. 3C); as inflammation is known to increase endothelial cell production of galectin-1 [11], 407

we speculate that, in vivo, galectin-1 produced by endothelial cells may increase target cell infection by 408

increasing viral attachment to host target cells. However, galectin-1 can also inhibit syncytia formation 409

among endothelial cells infected with NiV in vitro (Fig. 4). This effect may limit the extent or severity 410

of NiV pathogenicity in vivo, potentially reducing endothelial cell damage and the resulting 411

hemorrhagic diathesis. Moreover, the opposing effects of increased viral entry by facilitating viral 412

attachment without affecting viral fusion with the endothelial cell membrane versus inhibition of 413

membrane fusion during syncytia formation of infected cells suggests that these two types of fusion 414

events are not identical. 415

It is remarkable that complex N-glycans on a specific site in NiV-F (F3) can account for the 416

majority of galectin-1’s effect. To gain further insight into how galectin-1 binding to F3 N-glycan 417

mediates its dichotomous role in NiV infection and spread, we modeled a trimeric spike with the 418

cognate N-glycans at the relvant sites. Fig 5A shows that the F3 N-glycan (red) is positioned right 419


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“above” the fusion peptide (blue), and proximal to the putative target cell membrane. The distance 420

between glycan binding sites on the dimeric galectin-1 is ~50Å (Fig. 5B), which is too short to span 421

the F3 N-glycans between any two F protomers on the same timeric NiV-F spike. Thus, galectin-1 422

likely binds to the F3 N-glycans on NiVF protomers from distinct F spikes, forming a glycoprotein 423

lattice that blocks the triggering of the fusion peptide (Fig. 5A, legend). We posit that glycoprotein 424

lattice formation predominates when galectin-1 binds to NiV-F expressed on infected cells, thereby 425

inhibiting syncytia formation [11]. However, on the virion surface that is tightly packed with envelope 426

glycoproteins, glycoprotein lattice formation may be less efficient, and the ability of galectin-1 to 427

bridge virus predominates. The latter can lead to an enhancement of infection when receptor binding 428

triggers fusion by the other virion-associated F spikes that are not bound by galectin-1. 429

As mentioned above, while endothelial cells express abundant galectins, inflammation in 430

general and viral infection specifically are known to significantly increase endothelial cell expression 431

of several galectins, including galectin-1. Moreover, as secreted galectins bind back to glycoproteins 432

on the plasma membrane as well as to glycans on extracellular matrix glycoproteins, galectins can be 433

highly concentrated at the cell surface. Thus, the galectin-1 concentration used in these studies may 434

reflect the physiologic concentration of galectin-1 encountered by a virus at the endothelial cell 435

membrane [48-51]. 436

This study further characterizes the complex interaction between galectin-1 and NiV. While 437

other lectins have been shown to have effects both beneficial and detrimental to the host in microbial 438

infections [20, 21, 36, 39], we report these opposing effects for a single virus, NiV, and a single lectin, 439

galectin-1, in the same host cell. These studies may help to explain the range of clinical outcomes of 440

NiV infection, as some patients recover fully after infection, while other patients succumb. Perhaps 441

differential expression of galectin-1 in different individuals at different time points during infection 442


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either promotes virus-endothelial cell interactions or prevents endothelial cell damage. The present 443

work suggests a complex spatiotemporal interplay between an immune regulator and a virus that may 444

ultimately determine the outcome of infection. 445

446

Acknowledgements 447

This study was supported by NIH grant R01AI060694 (to L.G.B. and B.L.) a Ruth L. Kirschstein 448

National Research Service Award T32HL69766 (to O.B.G.), NIH grant R01AI109022 (to H.A.C.), and 449

Medical Research Council Grant MR/L009528/1 (to T.A.B.). A.N.F. acknowledges partial support by 450

a grant from the NIAID/NIH through the Western Regional Center of Excellence for Biodefense and 451

Emerging Infectious Disease Research (U54 AI057156). 452


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453

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56. Lopez-Lucendo, M.F., D. Solis, S. Andre, J. Hirabayashi, K. Kasai, H. Kaltner, H.J. 620

Gabius, and A. Romero. 2004. Growth-regulatory human galectin-1: crystallographic 621

characterisation of the structural changes induced by single-site mutations and their impact on 622

the thermodynamics of ligand binding. J. Mol. Biol. 343:957-970. 623

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Figure Legends 627

Figure 1. Galectin-1 enhances infection of NiVpp in a carbohydrate binding dependent manner. 628

(A) Quantification of galectin-1 enhancement of NiVpp infection. NiVpp was titrated such that viral 629

inoculum that gave luciferase activity in the linear range at 24hpi was used. NiVpp was added to 630

monolayers of Vero cells, and virus entry in the absence (white bars) and presence (black bars) of 631

increasing concentrations of galectin-1 was quantified by measuring luciferase activity in infected cell 632

lysate 24 hpi as described in Experimental Procedures. Data from one of three replicate experiments 633

are presented as mean fold-increase (+/- SD of triplicate samples) over the virus only (no galectin-1) 634

condition. Significant p value determined by two-way ANOVA. (B) Galectin-1 enhancement of NiVpp 635

infection is carbohydrate binding dependent. NiVpp (white bars) and 10 M galectin-1 (black bars) 636

was added to Vero cells in the presence of 100mM lactose or 100mM sucrose. Data are presented as in 637

(A); mean +/- SD of triplicate samples from one of three replicate experiments is shown. (C) Galectin-638

1 added during spinoculation (at 4C) shows an increase in NiVpp infection on Vero cells. NiVpp 639

infection in the absence (white bars) or presence (black bars) of 10M galectin-1 added during or after 640

spinoculation, i.e. during or after attachment of virus to cells. Data are mean +/- SD of triplicate 641

samples from one of three replicate experiments. Significant p value determined by Student’s t test. 642

643

Figure 2. Galectin-1 enhances infection of NiVpp by bridging the virus to the cell through 644

binding of viral surface and cell surface complex N-glycans. Flow cytometric analysis of L-PHA 645

binding to 293T cells (A) or Vero cells (C) treated with kifunensine (dashed black line) shows loss of 646

cell surface complex N-glycans when compared to L-PHA binding to untreated parental cells (bold 647

black outlined histograms). Gray filled histogram represents negative control with secondary only. 648

(B) Galectin-1 mediated enhancement of NiVpp infection is dependent upon complex N-glycans found 649


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on the surface of the virus. Virus infection of Vero cells in the absence (white) or presence of 20M 650

galectin-1 (black) is shown for NiVpp bearing three types of envelope glycoproteins; wild type NiV-F 651

+ -G, NiV-F3 mutant (+ wild type G), or NiV-F + -G devoid of complex N-glycans. Data from one of 652

three replicate experiments are presented as mean fold-increase in infection (+/- SD of triplicate 653

samples) over the virus only (no galectin-1) condition. (D) Galectin-1 mediated enhancement of 654

NiVpp infection is dependent upon complex N-glycans on the surface of Vero cells. Virus infection in 655

the absence (white bars) or presence (black bars) of 20 M galectin-1 is shown for wild-type Vero 656

cells and Vero cells devoid of complex N-glycans. Data (mean +/- SD of triplicate samples) from one 657

of three replicate experiments are presented exactly as described for (B). 658

659

Figure 3. Galectin-1 enhances NiVpp and live Nipah virus infection of HUVECs. 660

(A) NiVpp was added to monolayers of HUVECS in the absence (white bar) and presence (black bars) 661

of 20M of galectin-1 for 1 hour, and infection was quantified by measuring Renilla luciferase activity 662

at 24hpi. Data are presented as mean fold-increase (+/- SD of triplicate samples) in infection over the 663

virus only (no galectin-1) condition. One of three replicate experiments is shown. (B) Quantification of 664

galectin-1 enhancement of recombinant GLuc reporter NiV (rNiV-GLuc) infection of HUVECs. (B) 665

rNiV-GLuc was added to monolayers of HUVECs in the absence (white bar) and presence (black bars) 666

of the indicated amounts of galectin-1 for 1 hour, and infection was quantified by measuring Gaussia 667

luciferase activity. Data are presented as mean fold-increase (+/- SD of triplicate samples) in infection 668

over the virus only (no galectin-1) condition. One of three replicate experiments is shown. (C) 669

Galectin-1 mediated enhancement of rNiV-GLuc infection is dependent upon complex N-glycans on 670

the surface of HUVECs and on the surface of the virus. Virus or HUVECs deficient in complex N-671

glycans were made in the presence of 20M Kifunensine (Virus Kif+ and Cells Kif+, respectively). 672


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rNiV-GLuc infection in the absence or presence of increasing concentrations of galectin-1 is shown for 673

wild type rNiV-GLuc and HUVECs (Virus wt/Cells wt, black bars), wild type rNiV-GLuc and 674

complex N-glycan deficient HUVECs (Virus wt/Cells Kif+, dark grey bars), complex N-glycan 675

deficient rNiV-GLuc and wild type HUVECs (Virus Kif+/Cells wt, light grey bars), and complex N-676

glycan deficient rNiV-GLuc and HUVECs (Virus kif+/Cells Kif+, white bars). Data represent the 677

average fold- increase in infection determined as in (B), and presented as mean +/- SD of triplicate 678

samples from one of three replicate experiments. 679

680

Figure 4. Galectin-1 can have opposing effects on Nipah virus production and syncytia formation 681

(A) Effect of galectin-1 added post-infection on the replicative spread of Nipah virus. HUVECs were 682

infected with NiVMAL for 1 hour at 37 C, washed to remove excess virus, and 20M galectin-1 (black 683

bars) or buffer (virus only, white bars) added to the media only post-infection. Viral titers (log scale) 684

were quantified by plaque assay after 0, 12, 24, and 36 hpi. (B) Syncytia formation induced by NiV 685

infection. HUVECs infected with live NiVMAL or NiVMAL F3 mutant virus in the absence (no galectin-686

1), presence of galectin-1 added before infection, or presence of galectin-1 added after infection. Cells 687

were fixed at 24 hpi and stained with DAPI to reveal nuclei. (C) Quantitation of Figure 4C. Nuclei per 688

syncytium per field were enumerated for the three separate conditions. 689

690

Figure 5. Modeling of the glycosylated pre-fusion trimeric NiV-F spike and galectin-1 691

(A) A model of the glycosylated NiV-F ectodomain in the prefusion state was created with the SWISS-692

MODEL server [52] using the structure of the parainfluenza virus 5 F protein in the metastable, 693

prefusion conformation (PDB accession number 2B9B) [53] as a template. The model of the NiV-F 694

ectodomain is shown in the surface representation, with each protomer colored a different shade of 695


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gray. For each protomer, structures of complex-type glycans [54] were placed at N-linked 696

glycosylation sites F2-F4 and oligomannose-type glycans [55] were placed at F5. Glycans at sites F2 697

and F4 are colored pink, F3 are colored red, and at F5 are colored green. Residues corresponding to the 698

putative fusion peptide (residues 103-128) are colored blue. The distance between equivalent F3 699

glycans is approximately 100Å. (B) Homo-dimeric crystal structure of C2S human galectin-1 in 700

complex with lactose (PDB accession number 1W60) [56]. Galectin-1 is shown as a surface 701

representation with the two protomers colored different shades of gray. -lactose is bound to both 702

protomers and is shown as pink sticks. The distance between equivalent binding sites is approximately 703

50Å. Structures and models were rendered with pymol (www.pymol.org). 704

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Documents

Timing of galectin-1 exposure differentially modulates Nipah virus