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. 2019 Jan 17;93(3):e01746-18.
doi: 10.1128/JVI.01746-18. Print 2019 Feb 1.

Restriction of Human Cytomegalovirus Infection by Galectin-9

Emily A Machala  1 , Selmir Avdic  1 , Lauren Stern  1 , Dirk M Zajonc  2   3 , Chris A Benedict  2 , Emily Blyth  4   5   6   7 , David J Gottlieb  4   5   6   7 , Allison Abendroth #  1 , Brian P McSharry #  1 , Barry Slobedman #  8
Affiliations

Restriction of Human Cytomegalovirus Infection by Galectin-9

Emily A Machala et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections.IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.

Keywords: cytomegalovirus; human herpesviruses; virus-host interactions.

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Figures

FIG 1
FIG 1
Gal-9, but not Gal-1, inhibits HCMV infection. Merlin-GFP was treated with recombinant Gal-1 or Gal-9 for 30 min prior to infection of HFs (MOI, 0.5). Infection was assessed at 72 h p.i., with the percentage of GFP-positive cells quantified by flow cytometry. (A) Representative scatter plots of GFP gating. FSC, forward scatter. (B) Fold change in the percentage of infected cells during Gal-1 (12.5 to 100 nM) treatment is presented as mean + SEM (n = 3). (C) Fold change in the percentage of infected cells during Gal-9 (12.5 to 100 nM) treatment is presented as mean + SEM (n = 4). Statistical significance was determined by one-way ANOVA comparing to Merlin-GFP alone for each treatment group. (D) HFs were treated with Gal-9 (50 to 400 nM) for 90 min. At 24 h posttreatment, cell death was assessed using a Zombie-NIR stain and quantified by flow cytometry. Percentage of Zombie-NIR-negative cells (live) is presented as mean + SEM (n = 3). Statistical significance was determined by one-way ANOVA comparing to no treatment. TB40-GFP was treated with recombinant Gal-9 (25 to 200 nM) for 30 min prior to infection of HFs (MOI, 0.5) (E) or RPE-1 cells (MOI, 1) (F). Fold change in the percentage of infected cells during Gal-9 treatment was determined at 48 h p.i. and is presented as mean + SEM (HFs n = 3). Statistical significance was determined by one-way ANOVA comparing to TB40-GFP alone for each cell type (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).
FIG 2
FIG 2
Gal-9-mediated inhibition of HCMV infection can be blocked by anti-Gal-9 antibody and is carbohydrate recognition domain (CRD) dependent. (A) Gal-9 (50 nM) was blocked with an anti-Gal-9 antibody or isotype control for 30 min before treatment of HCMV (Merlin-GFP) for a further 30 min and infection of HFs (MOI, 0.5). Fold change in the percentage of infected cells at 72 h p.i. is presented as mean + SEM (n = 3). Statistical significance was determined by 2-way ANOVA comparing to HCMV for each treatment. Lactose or sucrose was used to block Gal-9 (50 nM) for 30 min before treatment of HCMV for a further 30 min and infection of HFs (MOI, 0.5). Infection was assessed at 72 h p.i. as the percentage of GFP-positive cells quantified by flow cytometry. (B) Fold change in the percentage of infected cells during lactose (1.25 to 5 mM) blocking is presented as mean + SEM (n = 4). (C) Fold change in the percentage of infected cells during sucrose (1.25 to 5 mM) blocking is presented as mean + SEM (n = 4). Statistical significance was determined by one-way ANOVA comparing to HCMV alone (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 3
FIG 3
Pretreatment of virus inoculum with Gal-9 inhibits HCMV infection. (A) Recombinant Gal-9 (50 nM) was used to pretreat HFs (cells) or HCMV (virus, Merlin-GFP) for 30 min before infection at an MOI of 0.5. (B) HFs were infected with HCMV at an MOI of 0.5. Following infection, Gal-9 was added for 24 h. Graphs show the fold change in the percentage of infected cells during Gal-9 treatment at 72 h p.i., presented as mean + SEM (n = 3). Statistical significance was determined by one-way ANOVA comparing to untreated HCMV (**, P < 0.01; ns, not significant). (C) HFs were infected with HCMV (Merlin-GFP) at an MOI of 0.001 (150 PFU) without or with Gal-9 (100 nM) pretreatment for 30 min. Virus was incubated with HFs for 90 min and then washed with PBS to remove nonadhered virus (C) or citrate buffer to inactivate noninternalized virus (D). Infections were performed in duplicate, with cells incubated in a semisolid overlay, and plaques were enumerated 12 days p.i. (PBS, n = 3; citrate buffer, n = 4). Plaque numbers are presented as mean + SEM, and statistical significance was determined using Student’s two-tailed t test (*, P < 0.05).
FIG 4
FIG 4
Gal-9 treatment inhibits HCMV early during infection of HFs. HFs were seeded onto coverslips and infected with HCMV (Merlin, MOI, 0.5) without or with 30 min of Gal-9 (100 nM) pretreatment. At 24, 48, and 72 h p.i., cells were fixed and stained for IE1 antigen or appropriate isotype control (mIgG2a), and staining was visualized using anti-mouse IgG Alexa Fluor 594-conjugated secondary antibody (red). Cells were counterstained with the nuclear dye DAPI (blue). (A) Cells were imaged using a wide-field fluorescent light microscope, and representative staining at 24 h p.i. is shown. (B) From the immunofluorescent images captured, the percentage of total cells that were IE1 positive was determined using ImageJ software. The percentage of IE1-positive cells is expressed as mean + SEM, from three independent experiments, with five representative images taken per experiment. Statistical significance was determined by 2-way ANOVA comparing to HCMV for each time point (*, P < 0.05; **, P < 0.01). (C) HFs were infected with HCMV (Merlin, MOI, 0.001) with or without 30 min Gal-9 treatment (100 nM). Cells were harvested at 4 h p.i. and extracted DNA quantified by qPCR. Viral DNA was quantified relative to cellular DNA and normalized to untreated HCMV infection. Relative viral DNA is presented as mean + SEM (n = 3). Statistical significance was determined by one-tailed Student’s t test (*, P < 0.05).
FIG 5
FIG 5
Gal-9 inhibits entry of HCMV. (A) Schematic representation of inhibition of entry experiment. HCMV was added to HFs at 4°C for 90 min, enabling binding of the virions to cell surface receptors but not fusion. Recombinant Gal-9 (50 nM) was added, and the infection was transferred to 37°C to allow fusion to occur in the presence of recombinant Gal-9 for 60 min. Noninternalized virus was inactivated and removed by washing with citrate buffer. Infection was assessed by percentage of GFP-positive cells quantified by flow cytometry at 72 h p.i. (B) Fold change in the percentage of infected cells during Gal-9 treatment is presented as mean + SEM (n = 3). (C) Gal-9 was preblocked with lactose or sucrose for 30 min before addition during fusion of HCMV with HFs. Fold change in the percentage of infected cells during treatment is presented as mean + SEM (n = 3). Statistical significance was determined by 2-way ANOVA comparing to mock infection for each time point (*, P < 0.05; **, P < 0.01; ns, not significant).
FIG 6
FIG 6
Upregulation of soluble Gal-9 during natural HCMV reactivation in hematopoietic stem cell transplant (HSCT) recipients. Gal-9 concentrations (in nanograms per milliliter) were determined by ELISA in plasma isolated from HSCT patients in the first 100 days posttransplant. Patients were designated into one of three groups: (i) HCMV negative, (ii) HCMV-positive nonreactivators, or (iii) HCMV-positive reactivators. Plasma samples taken at the initial detection of HCMV reactivation (T1), the peak of reactivation (T2), and the control of reactivation (T3) and time-matched samples for nonreactivation patients. Compiled data from patients is presented as the mean + SEM of data from patients tested (n = 3). Significant differences in soluble Gal-9 concentration for HCMV-positive reactivation group determined by 2-way ANOVA with Tukey’s multiple-comparison test (**, P < 0.01; ***, P < 0.001).
FIG 7
FIG 7
Upregulation of soluble Gal-9 during experimental productive HCMV infection and interferon treatment. (A) HFs were mock infected or infected with HCMV (MOI, 0.5). Supernatants were harvested at 24, 48, 72, and 96 h p.i. (n = 4). (B) HFs were untreated or treated with recombinant IFN-β (62.5 U/ml) for 24, 48, 72, or 96 h and supernatants collected at each time point (n = 3). Gal-9 concentrations (nanograms per milliliter) were determined by ELISA and presented as mean + SEM. Statistical significance was determined by 2-way ANOVA comparing to mock for each time point (*, P < 0.05; ****, P < 0.0001).
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