JGV Papers in Press. Published July 10, 2014 as doi:10.1099/vir.0.

066837-0

Acute Hantavirus Infection Induces Galectin-3 Binding Protein

Jussi Hepojoki1,*, Tomas Strandin1, Udo Hetzel2, Tarja Sironen1, Jonas Klingström3,
Jussi Sane1, Satu Mäkelä4,5, Jukka Mustonen4,5, Seppo Meri6, Åke Lundkvist7, Olli
Vapalahti1,2,8, Hilkka Lankinen1 and Antti Vaheri1,8
1
Department of Virology, Peptide and Protein Laboratory and 6Department of Bacteriology
and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
2
Veterinary Pathology, Department of Veterinary Biosciences, Faculty of Veterinary
Medicine, University of Helsinki, Finland
3
Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska
University Hospital Huddinge, Stockholm, Sweden
4
Department of Internal Medicine, Tampere University Hospital and 5School of Medicine,
University of Tampere, Tampere, Finland
7
Swedish Institute for Communicable Disease Control, Solna, Sweden
8
Department of Virology and Immunology, HUSLAB, Hospital District of Helsinki and
Uusimaa, Finland

Running title: Gal-3BP in hantavirus infection

Contents category: Negative stranded RNA viruses

*Corresponding author
Mailing address: Department of Virology
Haartman Institute
P.O. Box 21
Haartmaninkatu 3
FI-00014 University of Helsinki
Finland
Phone: +358-9-19126608
Fax: +358-9-191 26491
E-mail: jussi.hepojoki@helsinki.fi

Word number in summary: 200

Word number in text (excluding summary): 5690
 Summary

Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to

man. Severe hantavirus infection is manifested by impairment of renal function, pulmonary

edema, and capillary leakage. Both innate and adaptive immune responses contribute to the

pathogenesis but the underlying mechanisms are not fully understood. Here we describe that

galectin-3 binding protein (Gal-3BP) is upregulated as a result of hantavirus infection both in

vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum and increased

Gal-3BP levels have been reported in chronic viral infections and in several types of cancer.

Our in vitro experiments show that while Vero E6 cells (African green monkey kidney cell

line) constitutively express and secrete Gal-3BP, this protein was detected in primary human

cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of

cynomolgus macaques experimentally infected with hantavirus indicated that hantavirus

infection induces Gal-3BP also in vivo. Finally, the analysis of plasma samples collected

from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP

levels during the acute than convalescent phase. Furthermore, the Gal-3BP levels in HFRS

patients correlated with increased complement activation and with clinical variables

reflecting the severity of acute hantavirus infection.

 INTRODUCTION

Rodent and insectivore-borne hantaviruses comprise a genus in the family Bunyaviridae.

Hantaviruses are rodent- and insectivore-borne viruses that occasionally infect man causing

either hemorrhagic fever with renal syndrome (HFRS, Eurasian hantaviruses) or hantavirus

cardiopulmonary syndrome (HCPS, American hantaviruses) with respective case-fatality

rates of 0.1-12% and 30-40% (Vaheri et al., 2013). Severe cases of HFRS and HCPS are

manifested by impairment of renal function, pulmonary edema, and by vascular leakage. The

mechanisms underlying these clinical signs remain largely unknown. However, the

involvement of the complement system has been suggested (Sane et al., 2011). The main

HFRS-causing viruses include Hantaan, Dobrava, Seoul and Puumala virus (PUUV). HCPS

is mainly caused by Sin Nombre and Andes viruses (Jonsson et al., 2010). Tula virus (TULV)

is considered a low-virulent or apathogenic hantavirus with only a few reported cases

(Klempa et al., 2003).

PUUV-induced HFRS is clinically characterized by high fever, headache, nausea,

blurred vision, backache and abdominal pains. Proteinuria, hematuria, and oliguria, followed

by polyuria indicate renal dysfunction, which is typical in HFRS (Vaheri et al., 2013).

Common laboratory findings are leukocytosis, thrombocytopenia, anemia, and elevation of

plasma C-reactive protein (CRP) and creatinine levels. The clinical severity of PUUV-

infection varies greatly and host genes influence the clinical picture (Makela et al.,

2002,Mustonen et al., 1996). Complete recovery is the usual outcome. Generally, hantavirus

infection leads to intense immune activation associated with production of cytokines such as:

TNF-α, interleukin (IL)-1, IL-5, IL-6, IL-10, IL-15, interferon (IFN)-α, IFN-β, IFN-γ and

RANTES/CCL5 (Borges et al., 2006,Jonsson et al., 2010,Vaheri et al., 2011). The infection

also results in increased numbers of activated CD8+ T cells and natural killer (NK) cells

(Bjorkstrom et al., 2011,Tuuminen et al., 2007). Moreover, previous studies have shown that
the extent of complement activation via the alternative pathway in PUUV-infection is

common and associated with disease severity (Paakkala et al., 2000,Sane et al., 2011). In this

report we describe upregulation of galectin-3 binding protein (Gal-3BP, previously known as

Mac-2BP and also called as 90K) as a result of hantavirus infection. Upregulation of Gal-3BP

has earlier been reported in chronic viral infections (Artini et al., 1996,Kittl et al.,

2000,Longo et al., 1993,Natoli et al., 1994), but this is to our knowledge the first report of

Gal-3BP induction in acute virus infection.

 RESULTS

Induction of Gal-3BP by hantavirus infection in primary endothelial cells. Elevated

serum levels of Gal-3BP have been reported in HIV, HBV and HCV infections (Artini et al.,

1996,Natoli et al., 1994). Moreover, the transcription of Gal-3BP is according to microarray

analysis up-regulated in human umbilical vein endothelial cells (HUVEC) infected by

different hantaviruses (Geimonen et al., 2002). We wanted to study whether hantavirus

infection would induce the secretion of Gal-3BP in Vero E6 cells and analyzed the amount of

Gal-3BP released from TULV infected and mock-infected cells. High levels of Gal-3BP were

observed in supernatants from uninfected and TULV-infected cells, showing that Vero E6

cells both produce and secrete Gal-3BP independently of hantavirus infection (Fig. 1A).

Several cancer cells and other continuous cell lines overexpress Gal-3BP. Therefore, we

wanted to check whether infection of primary cells with pathogenic (PUUV) or apathogenic

(TULV and PHV) hantavirus, in contrast to the continuous Vero E6 cells, would induce Gal-

3BP production. We used real-time PCR to quantify the amount of Gal-3BP mRNA produced

in hantavirus-infected as compared to non-infected HUVECs. While mock-infected control

cells did not express Gal-3BP, the cells infected with PUUV, TULV, and Prospect Hill virus

(PHV) were found to express Gal-3BP (Fig. 1B). The upregulation of Gal-3BP was observed

with both apathogenic (TULV and PHV) and pathogenic (PUUV) hantaviruses. By using

immunoblotting, we could demonstrate that Gal-3BP upregulation occurred also at protein

level (Fig. 1C). To assess whether Gal-3BP would be secreted from infected cells, we

measured the Gal-3BP levels in the supernatants by ELISA. As expected, the Gal-3BP

produced as a result of hantavirus infection was secreted to the growth medium (Fig. 1D).

Finally, we wanted to study whether Gal-3BP up-regulation would affect virus

replication, and used commercially available RNAi to knock-down Gal-3BP expression in

HUVECs. Transfection of TULV-infected cells with Gal-3BP-specific siRNA duplexes #1

and #2 at 5 nM concentration successfully knocked-down Gal-3BP induced by TULV (Fig.

1E). Only a slight decrease in Gal-3BP level was observed with the siRNA duplex #3,

whereas TULV-infected cells transfected with scrambled control siRNA were found to

produce Gal-3BP. The knock-down of Gal-3BP (Fig. 1E, lanes 2 and 3) resulted in increased

amount of viral N protein, indicating that Gal-3BP expression negatively affects the virus

replication.

Upregulation of Gal-3BP production as a result of PUUV infection in cynomolgus

macaques. The observation that hantaviruses induce Gal-3BP in human endothelial cells, the

primary target of hantavirus infection in vivo, prompted us to study whether upregulation of

Gal-3BP would be observed in hantavirus-infected animals. In a previous study cynomolgus

macaques experimentally infected with PUUV showed symptoms mimicking human PUUV

infection (Klingstrom et al., 2002,Klingstrom et al., 2008). We set up an in-house capture

ELISA for the detection of Gal-3BP from serum samples of cynomolgus macaques, and used

the test to determine the GAL-3BP concentrations at different time points of the archival

experimental infection samples (Klingstrom et al., 2002,Klingstrom et al., 2008).

Upregulation of Gal-3BP was seen in 5/6 of animals challenged with PUUV (Fig. 2A animals

#4040, #4118 and #12040 in left panel; animals #53 and #59 in right panel). Gal-3BP

remained at low level in passively immunized animals (Fig. 2A, animals #2190, #6114 and

#11298 in left panel) even after challenge with PUUV. The upregulation of Gal-3BP was

observed at around 2 weeks after inoculation (14 or 15 d). The exact kinetics of Gal-3BP

induction, however, could not be determined due to limited number of time points in the

original study. We also correlated serum Gal-3BP levels to other variables measured earlier

from these samples (Klingstrom et al., 2002,Klingstrom et al., 2008). Serum Gal-3BP level

correlated positively with serum creatinine, TNF-α, IgM and IL-10 levels (Table 1).
Statistically significant negative correlation was observed between nitric oxide and Gal-3BP

level.

The level of Gal-3BP in plasmas of humans with acute PUUV infection. As we

observed upregulation of Gal-3BP in the serum samples of animals experimentally infected

with PUUV, we decided to measure the level of Gal-3BP in the plasma samples of patients

with acute HFRS. For this purpose we used a panel of well-characterized plasma samples

consecutively collected from patients hospitalized due to acute PUUV infection. The

maximum plasma level of Gal-3BP during hospitalization was used for the calculation of

correlations to other variables previously measured from aliquots of the same samples

(Paakkala et al., 2000,Sane et al., 2011). The median maximum plasma Gal-3BP-level in the

acute stage of PUUV infection (7.7 µg/ml, range 2.1-19.3 µg/ml) was found to be

significantly higher (p<0.001) than the level measured at convalescence (3.9 µg/ml, range

1.4-10.2 µg/ml), Fig 2B. The maximum plasma level of Gal-3BP correlated positively with

the treatment time at hospital (r=0.349, P=0.007), and with variables reflecting the fluid

retention during acute infection, such as change in weight during the hospital stay (r=0.321,

P=0.013), the highest urinary output in the polyuric phase (r=0.307, P=0.020), and the highest

systolic and diastolic blood pressure (r=0.339, P=0.009 and r=0.401, P=0.002, respectively).

The maximum plasma level of Gal-3BP correlated significantly also with the maximum level

of serum terminal complement complex SC5b-9 (r=0.409, P=0.002) and the maximum level

of plasma complement factor C4 (r=0.342, P=0.009). Maximum plasma creatinine levels

correlated slightly, but not statistically significantly with Gal-3BP levels (r=0.246, P=0.060).

The maximum Gal-3BP level did not correlate with the maximum blood leukocyte or

minimum platelet count, with minimum levels of plasma C3 or C4, nor with the maximum

plasma CRP and IL-6 level. The correlations of Gal-3BP level with other parameters

measured from the same samples are summarized in Table 2.

 Increased levels of Gal-3BP are observed in tissues that are positive for viral

antigen. Finally, we wanted to investigate whether Gal-3BP is upregulated in tissues of

infected animals. We stained lung, kidney, spleen, and liver preparations from PUUV-

infected cynomolgus macaques by immunohistochemistry, tissues collected from uninfected

animals acting as controls. These tissues have previously been stained for the presence of

viral nucleoprotein (Sironen et al., 2008). Prominent, infection-associated, Gal-3BP

expression was seen in the bronchial and bronchiolar respiratory epithelium (Fig. 3A), in

pulmonary alveolar lining cells (Fig. 3C, pneumocytes and alveolar macrophages), and in

lymphocytes in the bronchus-associated lymphatic tissue (BALT) (Fig. 3B). In the kidneys,

glomerular mesangial cells and the tubular epithelium exhibited strong Gal-3BP staining (Fig.

3D). In spleen (Fig. 3E) and liver (Fig. 3F), no positive reaction was observed in two animals

(a25, a53), whereas a positive reaction was seen in follicular lymphocytes in the spleen and

liver of animal #59. Since the staining was undertaken on tissues collected 4 weeks post

infection, at a time point when clinical signs were no longer observed, it was not possible to

evaluate Gal-3BP expression during the acute phase of infection. The tissues of uninfected

control animals did not show any reaction (Fig. 3G-J).

 DISCUSSION

Here we report that hantavirus infection induces Gal-3BP production. Initially, in vitro

experiments demonstrated that Vero E6 cells (a continuous simian cell line) produce Gal-3BP

irrespective of hantavirus infection, however, primary human cells (HUVECs) were found to

produce Gal-3BP only after hantavirus infection. The production of Gal-3BP was

accompanied by decreased amount of viral N protein, indicating that Gal-3BP negatively

affects hantavirus infection. Detection of increased Gal-3BP levels in the sera of animals

(cynomolgus macaques) experimentally infected with PUUV, and in the plasmas of patients

hospitalized due to acute PUUV infection indicated that hantaviruses induce Gal-3BP also in

vivo. The Gal-3BP levels were found to correlate with the levels of acute phase proteins (in

the case of animals), and with complement activation and clinical severity of PUUV infection

(in the case of human patients). Furthermore, upregulation of Gal-3BP was also seen in the

tissues of cynomolgus macaques infected with PUUV.

Although increased serum levels of Gal-3BP have been reported in several pathological

conditions, ranging from chronic viral infections to cancer, no unambiguous function has

been assigned to this protein (Artini et al., 1996,Darcissac et al., 2001,Grassadonia et al.,

2004). Recently, Gal-3BP was reported to interact with and aggregate recombinant adeno-

associated virus type 6 (Denard et al., 2012), however, the function of this interaction

remains to be studied. Our unpublished results show that both apathogenic and pathogenic

hantaviruses (TULV and PUUV) interact with Gal-3BP. Increased Gal-3BP levels are

reported in chronic viral infections of HIV, HBV and HCV; in the case of HIV the level of

Gal-3BP has been shown to correlate with the viral load (Groschel et al., 2000). Fairly

recently Gal-3BP expression was demonstrated to impair the infectivity of HIV (Lodermeyer

et al., 2013), indicating that Gal-3BP may act as an effector of antiviral response. However,

no direct interaction between Gal-3BP and HIV has been demonstrated, and the reduction of
HIV infectivity by Gal-3BP was shown to be mediated via interfering with the particle

maturation (Lodermeyer et al., 2013). The finding that hantavirus-induced Gal-3BP interferes

with the accumulation of N protein in endothelial cells suggests Gal-3BP to be involved in

the cellular antiviral response also against hantaviruses. While the mechanism is still

unresolved it could be due to the ability of Gal-3BP to bind hantaviruses directly. This could

take place either in the extracellular space where Gal-3BP might inhibit viral spread or in

intracellular compartments where it could block virus maturation as in the case of HIV-1

(Lodermeyer et al., 2013). On the other hand, the binding of Gal-3BP to viruses could act as

an alternative mode of antiviral action via triggering innate immune response by activation of

the complement system. In support of this hypothesis we observed a correlation between

complement activation and Gal-3BP production in the plasmas of patients hospitalized due to

acute PUUV infection.

On the other hand, Gal-3BP is known to induce the expression of cytokines such as IL-1,

IL-6, and TNF-α (Υλλριχη ετ αλ., 1994), the levels of which are also elevated during

hantavirus infection (Jonsson et al., 2010). High plasma IL-6 levels have been shown to

associate with the overall severity of acute PUUV infection (Outinen et al., 2010). In the

present study Gal-3BP levels were found to have a significant correlation with serum

creatinine level, TNF-α, IL-10 and nitric oxide level in the blood samples of monkeys

experimentally infected with PUUV. These findings indicate that Gal-3BP is indeed an acute-

phase protein and might act as an indicator for pathogenesis at cellular level. It seems likely

that Gal-3BP induction would not be specific for hantavirus infection, but would rather act as

a general trigger for immune activation in viral (or bacterial) infections. It is intriguing to

hypothesize that Gal-3BP would act as a sensor in endoplasmic reticulum (ER) and that it

might be able to recognize stress conditions of ER (due to, for example, overexpression of
viral proteins). In such conditions the secretion of Gal-3BP could increase, thus resulting in

the activation of an innate immune response.

The induction and presence of Gal-3BP could also directly contribute to the pathogenesis

of hantavirus infection since it has been shown to activate T and NK cells, both of which

have been shown to be activated in acute hantavirus-infection (Bjorkstrom et al., 2011). The

fact that Gal-3BP induces production of MMP-7 (Ulmer et al., 2010) could also be linked to

the pathogenesis of HFRS, since the substrates of MMP-7 include collagen IV and other

components of glomerular basement membranes (Thrailkill et al., 2009), whose degradation

would result in increased glomerular permeability. The induction of MMP-7 production and

subsequent degradation of e.g. VE-cadherin (Ichikawa et al., 2006) or release of vascular

endothelial growth factor (VEGF) (Ii et al., 2006) could, on the other hand, explain the

disruption of the endothelial barrier and resulting leakage of capillaries observed in HCPS

and HFRS (Gorbunova et al., 2010,Mackow & Gavrilovskaya., 2009,Shrivastava-Ranjan et

al., 2010). Curiously, Gal-3BP has recently been shown to induce VEGF production (Piccolo

et al., 2013), and elevated levels of VEGF were reported in pulmonary edema fluid of HCPS

patients (Gavrilovskaya et al., 2012) and in the sera of patients with acute HFRS (Li et al.,

2012). Interestingly, in the present study, the plasma Gal-3BP level correlated with several

variables reflecting fluid retention during acute PUUV infection. The upregulation of VEGF

in hantavirus-mediated diseases could thus be induced by Gal-3BP production.

 MATERIAL AND METHODS

Ethics statement. Written informed consent was obtained from all patients, and the

Ethics Committee of the Tampere University Hospital approved the study protocol. The

details of the experimental PUUV infections of cynomolgus macaques were described earlier

(Klingstrom et al., 2002,Klingstrom et al., 2008).

Cells, viruses and purification of viruses. Green monkey kidney epithelial Vero E6

cells (ATCC: CRL-1586) were grown and infected with multiplicity of infection (MOI) value

of 0.1-0.5 as described (Hepojoki et al., 2010a,Huiskonen et al., 2010). Human umbilical

vein endothelial cells, HUVECs, were grown in Endothelial Cell Growth Medium (MV,

PromoCell) containing supplement mix (Endothelial Growth Medium Supplement Mix,

PromoCell) with 40 µg/ml gentamicin and 250 ng/ml Fungizone in bottles pre-coated with

gelatin at 37 °C in a humidified atmosphere containing 5% CO2. Hantavirus infections on

HUVECs were done on 12-well plate with MOI value 1.

RNA interference (RNAi). The siRNA duplexes, LGALS3BP (ID3959) Trisilencer-27

Human siRNA (catalogue number SR302677), for in vitro knock-down of Gal-3BP were

obtained from Origene. RNAi was done with TULV-infected HUVECs on 12-well plate.

Briefly, cells were plated to 75-90% confluency, infected with MOI value 1, and transfected

with small interfering RNA (siRNA) duplexes (universal scrambled negative control and 3

Gal-3BP specific) according Lipofectamine RNAiMAX (Invitrogen by lifetechnologies)

protocol. At 3 days post infection and approximately 72 h post transfection the cells were

collected, and analyzed for Gal-3BP expression and virus production by immunoblotting.

After optimization 5 nM concentration of siRNA duplexes was found optimal for HUVEC

transfection.
 SDS-PAGE, immunoblotting and antibodies. SDS-PAGE separations were performed

according to standard protocols. Transfer of proteins on to nitrorocellulose was done by wet-

blotting. The probing of nitrocellulose membranes was done as described earlier (Hepojoki et

al., 2010b). The primary antibodies used were: Polyclonal anti-2/3N rabbit sera (Vapalahti et

al., 1992), polyclonal goat anti-human Gal-3BP antibody (R&D Systems), rabbit anti-Gal-

3BP serum and monoclonal anti-Gal-3BP antibody 12D4 (Laferte et al., 2000). The

secondary antibodies anti-rabbit IR800Dye and anti-goat AlexaFluor 680 (used in 1:10000)

were from Invitrogen.

Real-time PCR. A portion of mock-, PUUV-, TULV- and PHV-infected HUVECs used

in immunoblotting was stored for RNA isolation in Trisure (Bioline) reagent. Isolated RNAs

were reverse transcribed to cDNA with random hexamers and real-time PCR (Stratagene

MX3500P) of Gal-3BP was achieved using primer sequences described earlier (Park et al.,

2008). Relative quantification of Gal-3BP mRNA was done according to the 2-∆∆Ct method

(Livak & Schmittgen., 2001). The mRNA levels of RNA polymerase 2 were used as

reference (primer sequences for RNA polymerase 2 Forward: 5´-

GCACCACGTCCAATGACAT-3´ and Reverse 5´-GTGCGGCTGCTTCCATAA-3´).

Plasma assays of patients with acute PUUV infection. The patient material consisted

of consecutive plasma samples from 61 hospitalized patients (44 males and 17 females,

treated at the Tampere University Hospital during 2000-2004) with serologically confirmed

acute PUUV infection based on PUUV-IgM in µ-capture enzyme immunoassay (Vapalahti et

al., 1996). In median four samples per one patient were taken during hospital care and a

single sample for each patient was collected at the convalescent phase (roughly one month

after the onset of fever). The routine laboratory assays were determined at the Laboratory

Center of the Tampere University Hospital. The complement analysis was performed by

measuring SC5b-9 concentrations using an ELISA kit (Quidel, San Diego, CA) and Gal-3BP
levels in plasma were determined using Human Gal-3BP Platinum ELISA (Bender

MedSystems) according to manufacturer’s protocol.

Serum and tissue samples from PUUV infected cynomolgus macaques. The samples

used in this study were collected and analyzed for cytokines and acute-phase proteins during

experimental PUUV infection reported (Klingstrom et al., 2002,Klingstrom et al., 2008). The

tissue samples have previously been stained positive for PUUV N protein in

immunohistochemistry (Sironen et al., 2008).

ELISA assay for measuring of primate Gal-3BP. Purified IgG fraction of rabbit anti-

Gal-3BP was coated (100 µl/well diluted 1:100 in 0.1M NaHCO3, pH 9.3) to MaxiSorp™

(NUNC/Thermo Scientific) plates overnight at +4 ⁰C. After washing the wells with PBS-T

(PBS + 0.05% Tween 20) the plate was blocked for 45 min at +37 ⁰C with 2.5% BSA in PBS

(150 µl/well). The samples (diluted 1:100 in PBS with 2% BSA) and standards (from Human

Gal-3BP Platinum ELISA, Bender MedSystems) were applied in duplicate on plate (100

µl/well) followed by 45 min incubation at +37 ⁰C. After three PBS-T washes goat anti-human

Gal-3BP (R&D Systems) was added (100 µl/well, diluted 1:400 in PBS with 2% BSA)

followed by 45 min incubation at +37 ⁰C and three PBS-T washes. Rabbit anti-goat HRP

(Dakocytomation) was applied to wells (100 µl/well, diluted 1:1000 in PBS with 2% BSA)

and the plate was incubated for 45 min at +37 ⁰C. The detection after three PBS-T and a

single PBS wash was done using 3,3´,5,5´-Tetramethylbenzidine (TMB) liquid substrate

solution according to manufacturer’s recommendations (Sigma-Aldrich). The results were

read by Multiskan (Thermo Scientific) at 450 nm. The concentration of Gal-3BP in

supernatants of hantavirus infected HUVECs was determined using the above protocol.

Statistical analyses. The statistical analyses of the levels of factors in serum and plasma

samples were performed using SPSS software version 18. The maximum values of Gal-3BP
and SC5b-9 during the acute stage were used in the analyses. Correlations between the

variables were analyzed with Spearman’s rank correlation test. All p-values relate to two-

tailed tests and statistical significance was considered at 5% level.

Immunohistochemistry. Samples of lung, kidney, spleen, and liver were dissected,

fixed in 3% paraformaldehyde for 48 hours, dehydrated and paraffin-embedded. Sections of 4

µm were cut on slides prior to the staining that was performed on a fully automated Ventana

Discovery Slide Stainer (Ventana Medical Systems). The Gal-3BP specific MAb 12D4 was

used to visualize Gal-3BP in tissue samples of mock and PUUV infected monkeys. Ventana

IHC DAB MAP kit was used for detection, and sections were counterstained with

hematoxylin and postcounterstained with Bluing Reagent. Finally, the slides were rinsed and

dehydrated before mounting with EuKitt medium.

 ACKNOWLEDGEMENTS

Ms. Irina Suomalainen is thanked for skilful assistance in immunohistochemical staining

experiments done using VENTANA. Prof. Suzanne Laferté and Prof. William G. Chaney are

acknowledged for providing anti-Gal-3BP antiserum. This work was supported by the

following grants: European Commission Project [grants QLK2-CT-2002-01358, GOCE-CT-

2003-010284 EDEN]; and the Academy of Finland [grant number 259376]; and Sigrid

Jusélius Foundation [grant number 39-3554-30]; Helsinki University Hospital Research

Funds; and the Competitive State Research Funding of the Expert Responsibility Area of

Tampere University Hospital [grant number 9P031]; and Tampere Tuberculosis Foundation;

and Finnish Kidney Foundation; and Orion-Farmos Research Foundation; and Magnus

Ehrnrooth Foundation.
 REFERENCES

Artini M., Natoli C., Tinari N., Costanzo A., Marinelli R., Balsano C., Porcari P.,

Angelucci D., D'Egidio M., Levrero M., Iacobelli S. (1996). Elevated serum levels of

90K/MAC-2 BP predict unresponsiveness to alpha-interferon therapy in chronic HCV

hepatitis patients. J Hepatol 25, 212-217.

Bjorkstrom N. K., Lindgren T., Stoltz M., Fauriat C., Braun M., Evander M.,

Michaelsson J., Malmberg K. J., Klingstrom J., Ahlm C., Ljunggren H. G. (2011).

Rapid expansion and long-term persistence of elevated NK cell numbers in humans

infected with hantavirus. J Exp Med 208, 13-21.

Borges A. A., Campos G. M., Moreli M. L., Souza R. L., Aquino V. H., Saggioro F. P.,

Figueiredo L. T. (2006). Hantavirus cardiopulmonary syndrome: Immune response and

pathogenesis. Microbes Infect 8, 2324-2330.

Darcissac E. C., Vidal V., De La Tribonniere X., Mouton Y., Bahr G. M. (2001).

Variations in serum IL-7 and 90K/Mac-2 binding protein (mac-2 BP) levels analysed in

cohorts of HIV-1 patients and correlated with clinical changes following antiretroviral

therapy. Clin Exp Immunol 126, 287-294.

Denard J., Beley C., Kotin R., Lai-Kuen R., Blot S., Leh H., Asokan A., Samulski R. J.,

Moullier P.& other authors. (2012). Human galectin 3 binding protein interacts with

recombinant adeno-associated virus type 6. J Virol 86, 6620-6631.

Gavrilovskaya I., Gorbunova E., Koster F., Mackow E. (2012). Elevated VEGF levels in

pulmonary edema fluid and PBMCs from patients with acute hantavirus pulmonary

syndrome. Adv Virol 2012, 674360.

Geimonen E., Neff S., Raymond T., Kocer S. S., Gavrilovskaya I. N., Mackow E. R.

(2002). Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial

cell responses. Proc Natl Acad Sci U S A 99, 13837-13842.

Gorbunova E., Gavrilovskaya I. N., Mackow E. R. (2010). Pathogenic hantaviruses

andes virus and hantaan virus induce adherens junction disassembly by directing

vascular endothelial cadherin internalization in human endothelial cells. J Virol 84,

7405-7411.

Grassadonia A., Tinari N., Iurisci I., Piccolo E., Cumashi A., Innominato P., D'Egidio

M., Natoli C., Piantelli M., Iacobelli S. (2004). 90K (mac-2 BP) and galectins in tumor

progression and metastasis. Glycoconj J 19, 551-556.

Groschel B., Braner J. J., Funk M., Linde R., Doerr H. W., Cinatl J.,Jr, Iacobelli S.

(2000). Elevated plasma levels of 90K (mac-2 BP) immunostimulatory glycoprotein in

HIV-1-infected children. J Clin Immunol 20, 117-122.

Hepojoki J., Strandin T., Vaheri A., Lankinen H. (2010a). Interactions and

oligomerization of hantavirus glycoproteins. J Virol 84, 227-242.

Hepojoki J., Strandin T., Wang H., Vapalahti O., Vaheri A., Lankinen H. (2010b).

Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein. J

Gen Virol 91, 2341-2350.

Huiskonen J. T., Hepojoki J., Laurinmaki P., Vaheri A., Lankinen H., Butcher S. J.,

Grunewald K. (2010). Electron cryotomography of tula hantavirus suggests a unique

assembly paradigm for enveloped viruses. J Virol 84, 4889-4897.

Ichikawa Y., Ishikawa T., Momiyama N., Kamiyama M., Sakurada H., Matsuyama R.,

Hasegawa S., Chishima T., Hamaguchi Y.& other authors. (2006). Matrilysin (MMP-7)
degrades VE-cadherin and accelerates accumulation of beta-catenin in the nucleus of

human umbilical vein endothelial cells. Oncol Rep 15, 311-315.

Ii M., Yamamoto H., Adachi Y., Maruyama Y., Shinomura Y. (2006). Role of matrix

metalloproteinase-7 (matrilysin) in human cancer invasion, apoptosis, growth, and

angiogenesis. Exp Biol Med (Maywood) 231, 20-27.

Jonsson C. B., Figueiredo L. T., Vapalahti O. (2010). A global perspective on hantavirus

ecology, epidemiology, and disease. Clin Microbiol Rev 23, 412-441.

Kittl E. M., Hofmann J., Hartmann G., Sebesta C., Beer F., Bauer K., Huber K. R.

(2000). Serum protein 90K/Mac-2BP is an independent predictor of disease severity

during hepatitis C virus infection. Clin Chem Lab Med 38, 205-208.

Klempa B., Meisel H., Rath S., Bartel J., Ulrich R., Kruger D. H. (2003). Occurrence of

renal and pulmonary syndrome in a region of northeast germany where tula hantavirus

circulates. J Clin Microbiol 41, 4894-4897.

Klingstrom J., Plyusnin A., Vaheri A., Lundkvist A. (2002). Wild-type puumala

hantavirus infection induces cytokines, C-reactive protein, creatinine, and nitric oxide

in cynomolgus macaques. J Virol 76, 444-449.

Klingstrom J., Stoltz M., Hardestam J., Ahlm C., Lundkvist A. (2008). Passive

immunization protects cynomolgus macaques against puumala hantavirus challenge.

Antivir Ther 13, 125-133.

Laferte S., Loh L. C., Keeler V. (2000). Monoclonal antibodies specific for human

tumor-associated antigen 90K/Mac-2 binding protein: Tools to examine protein

conformation and function. J Cell Biochem 77, 540-559.

Li Y., Wang W., Wang J. P., Pan L., Zhang Y., Yu H. T., Jiang W., Wang P. Z., Bai X.

F. (2012). Elevated vascular endothelial growth factor levels induce hyperpermeability

of endothelial cells in hantavirus infection. J Int Med Res 40, 1812-1821.

Livak K. J. & Schmittgen T. D. (2001). Analysis of relative gene expression data using

real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25, 402-408.

Lodermeyer V., Suhr K., Schrott N., Kolbe C., Sturzel C. M., Krnavek D., Munch J.,

Dietz C., Waldmann T., Kirchhoff F., Goffinet C. (2013). 90K, an interferon-stimulated

gene product, reduces the infectivity of HIV-1. Retrovirology 10, 111.

Longo G., Natoli C., Rafanelli D., Tinari N., Morfini M., Rossi-Ferrini P., D'Ostilio N.,

Iacobelli S. (1993). Prognostic value of a novel circulating serum 90K antigen in HIV-

infected haemophilia patients. Br J Haematol 85, 207-209.

Mackow E. R. & Gavrilovskaya I. N. (2009). Hantavirus regulation of endothelial cell

functions. Thromb Haemost 102, 1030-1041.

Makela S., Mustonen J., Ala-Houhala I., Hurme M., Partanen J., Vapalahti O., Vaheri

A., Pasternack A. (2002). Human leukocyte antigen-B8-DR3 is a more important risk

factor for severe puumala hantavirus infection than the tumor necrosis factor-alpha(-

308) G/A polymorphism. J Infect Dis 186, 843-846.

Mustonen J., Partanen J., Kanerva M., Pietila K., Vapalahti O., Pasternack A., Vaheri

A. (1996). Genetic susceptibility to severe course of nephropathia epidemica caused by

puumala hantavirus. Kidney Int 49, 217-221.

Natoli C., Ortona L., Tamburrini E., Tinari N., Di Stefano P., D'Egidio M., Ghinelli F.,

Sighinolfi L., D'Ostilio N., Piazza M. (1994). Elevated serum levels of a 90,000 daltons
tumor-associated antigen in cancer and in infection by human immunodeficiency virus

(HIV). Anticancer Res 14, 1457-1460.

Outinen T. K., Makela S. M., Ala-Houhala I. O., Huhtala H. S., Hurme M., Paakkala A.

S., Porsti I. H., Syrjanen J. T., Mustonen J. T. (2010). The severity of puumala

hantavirus induced nephropathia epidemica can be better evaluated using plasma

interleukin-6 than C-reactive protein determinations. BMC Infect Dis 10, 132.

Paakkala A., Mustonen J., Viander M., Huhtala H., Pasternack A. (2000). Complement

activation in nephropathia epidemica caused by puumala hantavirus. Clin Nephrol 53,

424-431.

Park Y. P., Choi S. C., Kim B. Y., Kim J. T., Song E. Y., Kang S. H., Yoon D. Y., Paik S.

G., Kim K. D., Kim J. W., Lee H. G. (2008). Induction of mac-2BP by nerve growth

factor is regulated by the PI3K/Akt/NF-kappaB-dependent pathway in the HEK293 cell

line. BMB Rep 41, 784-789.

Piccolo E., Tinari N., Semeraro D., Traini S., Fichera I., Cumashi A., La Sorda R.,

Spinella F., Bagnato A.& other authors. (2013). LGALS3BP, lectin galactoside-binding

soluble 3 binding protein, induces vascular endothelial growth factor in human breast

cancer cells and promotes angiogenesis. J Mol Med (Berl) 91, 83-94.

Sane J., Laine O., Makela S., Paakkala A., Jarva H., Mustonen J., Vapalahti O., Meri

S., Vaheri A. (2011). Complement activation in puumala hantavirus infection correlates

with disease severity. Ann Med .

Shrivastava-Ranjan P., Rollin P. E., Spiropoulou C. F. (2010). Andes virus disrupts the

endothelial cell barrier by induction of vascular endothelial growth factor and

downregulation of VE-cadherin. J Virol 84, 11227-11234.

Sironen T., Klingstrom J., Vaheri A., Andersson L. C., Lundkvist A., Plyusnin A.

(2008). Pathology of puumala hantavirus infection in macaques. PLoS One 3, e3035.

Thrailkill K. M., Clay Bunn R., Fowlkes J. L. (2009). Matrix metalloproteinases: Their

potential role in the pathogenesis of diabetic nephropathy. Endocrine 35, 1-10.

Tuuminen T., Kekalainen E., Makela S., Ala-Houhala I., Ennis F. A., Hedman K.,

Mustonen J., Vaheri A., Arstila T. P. (2007). Human CD8+ T cell memory generation in

puumala hantavirus infection occurs after the acute phase and is associated with

boosting of EBV-specific CD8+ memory T cells. J Immunol 179, 1988-1995.

Ullrich A., Sures I., D'Egidio M., Jallal B., Powell T. J., Herbst R., Dreps A., Azam M.,

Rubinstein M., Natoli C. (1994). The secreted tumor-associated antigen 90K is a potent

immune stimulator. J Biol Chem 269, 18401-18407.

Ulmer T. A., Keeler V., Andre S., Gabius H. J., Loh L., Laferte S. (2010). The tumor-

associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells

enhances extracellular levels of promatrilysin and is a novel substrate of matrix

metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage.

Biochim Biophys Acta 1800, 336-343.

Vaheri A., Mills J. N., Spiropoulou C. F., Hjelle B. (2011). Hantaviruses. In Zoonoses -

Biology, Clinical Practice and Public Health, 2nd edn, pp. 307-307-322. Edited by S. R.

Palmer, L. Soulsby, D. Brown & P. Torgeson. Oxford: Oxford University Press.

Vaheri A., Strandin T., Hepojoki J., Sironen T., Henttonen H., Makela S., Mustonen J.

(2013). Uncovering the mysteries of hantavirus infections. Nat Rev Microbiol 11, 539-

550.
Vapalahti O., Kallio-Kokko H., Salonen E. M., Brummer-Korvenkontio M., Vaheri A.

(1992). Cloning and sequencing of puumala virus sotkamo strain S and M RNA

segments: Evidence for strain variation in hantaviruses and expression of the

nucleocapsid protein. J Gen Virol 73 ( Pt 4), 829-838.

Vapalahti O., Lundkvist A., Kallio-Kokko H., Paukku K., Julkunen I., Lankinen H.,

Vaheri A. (1996). Antigenic properties and diagnostic potential of puumala virus

nucleocapsid protein expressed in insect cells. J Clin Microbiol 34, 119-125.

TABLE 1. Correlations of Gal-3BP level with variables previously measured from serum

samples of cynomolgus macaques experimentally infected with PUUV. Spearman’s rank

correlation test.

90K/Mac-2BP
Spearman's rho p-value (2-tailed) N
Creatinine .601 ,000 67
TNF-α .524 .000 61
IgM .507 .002 34
IL-10 .396 .002 61
Nitric oxide (NO) -.270 .027 67
Focus reduction neutralization test .320 .065 34
IFN-γ .305 .122 27
IL-6 .133 .307 61
CRP .113 .361 67

Abbreviations: TNF-α = tumor necrosis factor alpha, IgM = immunoglobulin M, IL =

interleukin, IFN-γ = interferon gamma, CRP = C-reactive protein.

TABLE 2. Correlations between maximum plasma Gal-3BP levels and variables reflecting

the severity of acute infection in 61 patients hospitalized due to acute PUUV infection.

Spearman’s rank correlation test.

90K/Mac-2BP (max)
Spearman's rho p-value (2-tailed) N
.349 .007 59
Duration of hospital stay
.321 .013 59
Change in body weight during hospital stay
.339 .009 58
Systolic blood pressure max
.401 .002 58
Diastolic blood pressure max
.307 .020 59
Daily urinary output max
.409 .002 57
Plasma TCC max
.342 .009 58
Plasma C4 max
.181 .173 58
Plasma C4 min
.246 .060 59
Plasma creatinine max
.166 .208 59
Plasma CRP max
.163 .274 47
Plasma IL-6 max

Abbreviations: Max = maximum, min = minimum, TCC = terminal complement complex,

CRP = C-reactive protein, IL-6 = interleukin-6.

 FIGURE LEGENDS

FIG. 1. Production of Gal-3BP in Vero E6 cells and induction of Gal-3BP in HUVECs. A) A

250 µl aliquot of supernatant from growth medium of mock- and TULV-infected Vero E6

cells was collected daily up to 6 dpi. The amount of Gal-3BP in the medium was quantified

using capture ELISA. B) Quantification of Gal-3BP mRNA in HUVECs by real-time PCR.

The results are shown as fold increase as compared to mock-infected cells. C) Mock-, TULV,

PUUV- and PHV-infected HUVECs were collected at 2, 3, and 4 dpi, proteins separated on

SDS-PAGE and sequentially immunoblotted with Gal-3BP-, hantavirus N protein- and actin-

specific antibodies. The detection was done using Odyssey Infrared Imaging System (LI-

COR) using IR800-conjugated secondary antibody for anti-N, and Alexa Fluor 680-

conjugated secondary antibodies for both anti-Gal-3BP polyclonal and anti-actin monoclonal.

D) Gal-3BP concentration in growth medium collected from mock-, TULV, PUUV- and

PHV-infected HUVECs. The amount of Gal-3BP in growth media collected at 2, 3, and 4dpi

was measured using in-house capture ELISA test. E) Knock-down of Gal-3BP in TULV-

infected HUVECs by RNAi. TULV-infected cells were transfected with control, Gal-3BP #1,

Gal-3BP #2 or Gal-3BP #3 siRNA duplexes at 5 nM. At 3 days post infection and

approximately 72 h post transfection the cells were collected, and analyzed for Gal-3BP

expression and virus production by immunoblotting as described above. MOI of 0.1-0.3 and 1

were used for experiments with Vero E6 and HUVEC experiments, respectively.

FIG. 2. Gal-3BP is produced in the acute phase of PUUV infection. A) Serum samples

sequentially collected from cynomolgus macaques challenged with PUUV were analyzed for

Gal-3BP using capture ELISA. Animals #2190, #6114 and #11298 (left panel) were protected

for PUUV infection by passive immunization with pooled sera collected from animals #25,

#53 and #59 (right panel). B) Paired plasma levels of Gal-3BP in acute- and covalescent-

phase samples from patients with PUUV-induced HFRS. The maximum value of plasma Gal-
3BP concentration during hospitalization was plotted with the convalescent-phase sample for

the same individual using PASW Statistics 18.

FIG. 3. Immunohistochemical staining of lung, kidney, spleen and liver samples from

cynomolgus macaques with anti-Gal-3BP MAb. A-C. Lungs. A. Overview, depicting the

Gal-3BP staining in bronchial and bronchiolar epithelial cells (arrows). B. Gal-3BP staining

in lymphocytes of the BALT (arrowheads) and bronchiolar epithelia (arrows). C. Pulmonary

alveolar lining cells (arrows, pneumocytes and alveolar macrophages) also exhibit a positive

reaction. D. Kidneys: Gal-3BP staining is seen in glomerular mesangial cells (arrows) and

tubular epithelial cells (arrowheads). E, F. Spleen (E) and liver (F): Gal-3BP staining is seen

in follicular lymphocytes, hepatocytes and bile duct epithelia (animal #59). G-J. Controls:

Lung, kidney, spleen and liver from uninfected animals.

Figure 1
Figure 2
Figure 3