The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Dipanwita Mitra; Mohammad H Hasan; John T Bates; Michael A Bierdeman; Dallas R Ederer; Rinkuben C Parmar; Lauren A Fassero; Quntao Liang; Hong Qiu; Vaibhav Tiwari; Fuming Zhang; Robert J Linhardt; Joshua S Sharp; Lianchun Wang; Ritesh Tandon

doi:10.1371/journal.ppat.1009803

. 2021 Aug 5;17(8):e1009803. doi: 10.1371/journal.ppat.1009803

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Dipanwita Mitra ^1,^#, Mohammad H Hasan ^1,^#, John T Bates ^1,², Michael A Bierdeman ², Dallas R Ederer ¹, Rinkuben C Parmar ¹, Lauren A Fassero ¹, Quntao Liang ^3,⁴, Hong Qiu ⁵, Vaibhav Tiwari ⁶, Fuming Zhang ⁷, Robert J Linhardt ⁷, Joshua S Sharp ³, Lianchun Wang ⁸, Ritesh Tandon ^1,^2,^3,^*

Editor: Eain A Murphy⁹

¹Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America

²Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, United States of America

³Biomolecular Sciences, School of Pharmacy, University of Mississippi, Oxford, Mississippi, United States of America

⁴College of Biological Science and Engineering, University of Fuzhou, Fujian, China

⁵Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America

⁶Department of Microbiology and Immunology, Midwestern University, Downers Grove, Illinois, United States of America

⁷Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of America

⁸Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, United States of America

⁹State University of New York Upstate Medical University, UNITED STATES

The authors have declared that no competing interests exist.

^✉

* E-mail: rtandon@umc.edu

Contributed equally.

Roles

Dipanwita Mitra: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Mohammad H Hasan: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

John T Bates: Investigation, Methodology, Resources

Michael A Bierdeman: Investigation, Methodology, Resources

Dallas R Ederer: Investigation, Methodology

Rinkuben C Parmar: Investigation, Methodology

Lauren A Fassero: Investigation, Methodology

Quntao Liang: Investigation, Methodology, Resources

Hong Qiu: Investigation, Methodology

Vaibhav Tiwari: Methodology, Resources

Fuming Zhang: Investigation, Methodology, Resources

Robert J Linhardt: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing

Joshua S Sharp: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Lianchun Wang: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing

Ritesh Tandon: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Eain A Murphy: Editor

PMCID: PMC8384199 PMID: 34352038

Abstract

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

Author summary

Heparan sulfate (HS) is a linear polysaccharide (sugar) found in all animal tissues. It binds to a variety of protein ligands, including cytokines, chemokines, growth factors and morphogens and regulates a wide range of biological activities, including developmental processes, angiogenesis, blood coagulation, and tumor metastasis. The molecular diversity in HS chains generates unique binding sites for specific ligands and can offer preferential binding for a specific virus over other viruses or cellular ligands. In the current study human cytomegalovirus (CMV) was found to bind preferentially to uniquely sulfated and polymerized HS using its specific surface glycoprotein (gB). The HS mimics designed to bind gB inhibited CMV infection. The results were corroborated by parallel studies in mutant mouse cells as well as using protein-polysaccharide binding assays. Combined together, the data suggests that CMV preferentially attaches to uniquely modified HS and thus this virus-host interaction is amenable to targeting by specifically designed HS mimics.

Introduction

The heparan sulfate (HS) proteoglycans are present on most cell types and function as cellular attachment receptors for medically important viruses, including human immunodeficiency virus (HIV), hepatitis-C virus (HCV), human papillomavirus (HPV), Dengue virus (DENV) and the recently emerged SARS-CoV-2 [1–6]. In addition, virtually all human herpesviruses, with the possible exception of Epstein Barr virus, use HS as an initial co-receptor for entry [7]. The interaction between cell surface HS and virus envelope is the initial event in the complex process of virus entry. A successful virus entry involves downstream co-receptor interactions ultimately leading to fusion between the virus envelope and the cell membrane [8].

Herpesviruses and several other enveloped viruses enter the host cells using two distinct pathways: 1) A pH-independent pathway which involves the fusion of the virus envelope with the plasma membrane; and 2) A pH-dependent pathway that involves endocytosis of the virus particle [9]. In cells, where binding of virus to cell surface receptors induces endocytosis, the usual consequence is the acidification of the endosome, which ultimately triggers fusion between the virus envelope and endosomal membrane [7]. Interestingly, human cytomegalovirus (HCMV) entry mainly follows direct fusion at the cell surface in fibroblasts, while entry into other relevant cell types, such as endothelial cells, follows an endocytic route [10,11]. Different virus glycoprotein complexes are involved in each case; however, HS functions as the primary attachment receptor. Moreover, the presence of HS receptors are well documented in endosomal membranes and HS receptors likely play roles in intracellular virus trafficking [12–15].

The herpesvirus envelope is a lipid bilayer derived from host cell membranes in which most cellular proteins have been displaced by viral membrane proteins. For HCMV, at least twenty three different viral glycoproteins have been found to be associated with purified virion preparations [16]. For most herpesviruses, the conserved glycoprotein B (gB) is required for virus entry and it binds to cell surface molecules, including HS, which is present not only as a constituent of cell surface proteoglycans but also as a component of the extracellular matrix and basement membranes in organized tissues [7,17]. HCMV gB binds to HS resulting in virus attachment [18] similar to its counterparts in herpes simplex virus (HSV)-1 [17,19] and varicella-zoster virus (VZV) [20]. Subsequently, HCMV gB binds to cellular protein receptors such as EGFR [21], PDGFα [22], and integrins [23,24], which culminates in virus entry. Treatment of cells with soluble form of gB inhibits HCMV entry [25]. HCMV binding and infection are reduced by soluble heparin and HS, as well as in cells treated with heparinases or those unable to produce HS [26]. A better structural understanding of these inhibitions will pave the way to design effective antivirals that are highly specific as well as effective.

The synthesis of HS is a complex process involving multiple specialized enzymes and is initiated from a tetrasaccharide (GlcA-Gal-Gal-Xyl) that is attached to the core protein [27–29]. HS polymerase is responsible for building the polysaccharide backbone with a repeating unit of -GlcA-GlcNAc-. The backbone is then modified by N-deacetylase/N-sulfotransferase (NDST) responsible for N-deacetylation and N-sulfation of selected glucosamine residues, C5-epimerase responsible for epimerization of selected glucuronic moieties to iduronic acid, 2-O-sulfotransferase (Hs2st; 2-O-ST) responsible for 2-O-sulfation of selected iduronic acid residues, 6-O-sulfotransferase (Hs6st; 6-O-ST) for 6-O-sulfation and finally (but rarely) 3-O-sulfotransferases (Hs3st; 3-O-ST) responsible for 3-O-sulfation [30,31]. The substrate specificities of these biosynthetic enzymes dictate the structures of HS products, including sulfation levels, the contents of L-iduronic acid (IdoA) units and the size of the polysaccharides [30]. The location of the sulfo groups and IdoA in turn play a crucial role in determining the binding and functions of HS.

In the current study, we investigated the impact of specific sulfations as well as degree of polymerization in terms of numbers of monosaccharide units (dp) in HS chain on both human and mouse CMV infection and binding. Purified CMV extracellular virions preferentially bound strongly to the longer sulfated HS chains but not to the shorter unsulfated HS chains on a glycoarray. Glycosaminoglycans of different dp were derivatized from enoxaparin (ES, a low molecular weight heparin) and tested for their ability to inhibit CMV infection in cell culture. The results show that longer glycan chains are more efficient at reducing CMV titers in cells compared to shorter chain glycans. Also, the cell lines defective in expression of various sulfotransferases showed significantly reduced CMV entry and replication. Finally, purified glycoprotein B showed strong binding to heparin, and desulfated heparin analogs competed poorly with heparin for gB binding. Overall, these results indicate that CMV binding to cell surface glycans is dependent on branch length and sulfation pattern of HS.

Materials and methods

Preparation of glycosaminoglycans (GAGs) oligosaccharides

Glycosaminoglycans of different dp were fractionated from enoxaparin by Bio-Gel P-10 chromatography as previously described [32]. Briefly, 15 mg/mL enoxaparin sodium derived from porcine intestinal mucosa (Sanofi-Aventis U.S., Bridgewater, NJ) was applied to a Bio-Gel P-10 column (2.5X120 cm, Bio-Rad, Hercules CA) and eluted with 0.2 M NH₄HCO₃ at a flow rate of 14 ml/h. Elution of oligosaccharides was monitored by absorbance at 232 nm. NH₄HCO₃ was removed by heating in oven at 50°C for 24 h.

Preparation of the 6-O-desulfated Arixtra with MTSTFA

A detailed procedure on the preparation of 6-O-desulfated Arixtra was published previously [33]. Briefly, 4 mg of Arixtra was added to 10 volumes (w/w) of N-Methy-N-(trimethylsilyl)-trifluoroacetamide (MTSTFA, Sigma, ≥98.5%) and 100 volumes (v/w) of pyridine. The mixture was heated at 100°C for 30 min, then quickly cooled in an ice-bath, followed by extensive dialysis and freeze-drying. The sample was resuspended in 50% acetonitrile/water at a concentration of 30 μM for later LC-MS/MS analysis.

LC-MS/MS analysis

The 6-O-desulfated Arixtra (30 μM) was analyzed on a Thermo Orbitrap Fusion Tribrid (Thermo Fisher Scientific) coupled with an Ultimate 3000 Nano LC system (Dionex) using direct infusion. The flow rate was set to 1 μl/min. Mobile-phase was 50% acetonitrile. Nanoelectrospray voltage was set to 2.0 kV in negative ion mode. Full MS scan range was set to 200–2000 m/z at a resolution of 60,000, RF lens was 6%, and the automatic gain control (AGC) target was set to 2.0 × 10⁵. For the MS/MS scans, the resolution was set to 50,000, the precursor isolation width was 3 m/z units, and ions were fragmented by collision-induced dissociation (CID) at a normalized collision energy of 80%.

Cells

Mouse embryonic fibroblasts (MEF) and human foreskin fibroblasts (HFF) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Cellgro, Manassas, VA) containing 4.5 g/ml glucose, 10% fetal bovine serum (SAFC, Lenexa, KS), 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin (Cellgro, Manassas, VA) at 37°C with 5% CO₂. Mouse lung endothelial cells (WT, Hs3st1^-/-, Hs3st4^-/-, Hs3st1/4-double-knockout, Hs6st1^-/-, Hs6st2^-/-, and Hs6st1/2 double-knockout) were generated and reported in our recent study [34].

Virus

MCMV (strain K181) was grown in MEF cells, while HCMV (Towne strain derived from Towne-BAC) was grown on HFF cells. Virus stock was prepared in 3X autoclaved milk, sonicated 3 times and stored at −80°C. 3X autoclaved milk was prepared from Carnation (Nestle) instant nonfat dry milk powder. 10% milk was prepared in nano pure water, pH was adjusted to 7.0 and was autoclaved for 3 times. During infection, media was removed from the wells of cell culture plates and appropriately diluted virus stock was absorbed onto the cells in DMEM without serum. Cells were incubated for 1 hour with gentle shaking every 10 mins followed by washing 3X with PBS. Fresh complete medium was added and cells were incubated until the end point. For extracellular virus (ECV) purification, HFF were seeded in roller bottles, grown to confluency and infected with HCMV (Towne strain derived from Towne-BAC) at MOI of 0.01. Two days after 100% cytopathic effect was observed, infected cell medium was collected and centrifuged at low speed to pellet cellular debris, and the supernatant was transferred to new tubes and centrifuged at 20,000 g for 1 hour to pellet the ECV. This ECV pellet was re-suspended in phosphate buffer, sonicated to eliminate any aggregates, loaded over 15–50% continuous sucrose gradients and centrifuged in a SW-41 rotor at 39,000 RPM for 20 min. ECV bands were visualized in incandescent light and harvested by puncturing the sides of the centrifuge tubes. These bands were washed once with phosphate buffer, spun again and the final pellet resuspended in low salt phosphate buffer. An aliquot of the sample was used for assessment of initial quality of ECV by negative staining and transmission electron microscopy. Purified ECV were shipped on ice to Z biotech (Aurora, CO) for glycoarray binding analysis.

Virus infection and cell viability assay

HFF cells plated in 12 well tissue culture plates were grown to confluency and pretreated for 1h with 10 μM concentration of candidate HS and then infected with HCMV (Towne strain derived from Towne-BAC) at a multiplicity of infection (MOI) of 3.0 or mock- infected in the presence of candidate HS. Five hundred μl of fresh complete medium containing HS was added to the wells on day 3 and day 6. For virus GFP enumeration assay, the cells were harvested at 5 days post infection and total number of GFP foci in three replicate wells for each were counted. An average of >200 foci were present in mock-treated infected cells. At the designated time points, media was removed and cells were harvested by trypsinization. Cell viability was determined using trypan blue exclusion on TC20 automated cell counter (BioRad Laboratories, Hercules, CA) following manufacturer’s protocol.

Virus titers

Infected or mock-infected samples were harvested within the medium at the designated end points and stored at −80°C before titration. In some experiments, media and cells were separated by low-speed (< 1000 × g) centrifugation and viral loads in supernatant and cells were quantified by titering on wild-type cells. Titers were performed as described earlier [35] with some modifications. In brief, monolayers of fibroblasts grown in 12 well plates and serial dilutions of sonicated samples were absorbed onto them for 1 h, followed by 3X washing with PBS. Carboxymethylcellulose (CMC) (Catalog No. 217274, EMD Millipore Corp., Billerica, MA) overlay with complete DMEM media (1-part autoclaved CMC and 3 parts media) was added and cells were incubated for 5 to 10 days. At the end point, overlay was removed and cells were washed 2X with PBS. Infected monolayers were fixed in 100% methanol for 7 min, washed once with PBS and stained with 1% crystal violet (Catalog No. C581-25, Fisher Chemicals, Fair Lawn, NJ) for 15 min. Plates were finally washed with tap water, air dried and plaques with clear zone were quantified.

Immunoblots

Mouse lung endothelial cells (WT, Hs3st1^-/-, Hs3st4^-/-, Hs3st1/4-double-knockout, Hs6st1^-/-, Hs6st2^-/-, and Hs6st1/2 double-knockout) were infected with MCMV (K181 strain) and an MOI of 3.0 and the whole cell lysates were harvested at 2 hours post infection for analysis. The blot was probed with anti-IE1 antibody (catalog no. HR-MCMV-12, Center for Proteomics, University of Rijeka, Croatia) and HRP-conjugated goat anti mouse antibody (PI131444, Invitrogen) was used as the secondary antibody.

Glycoarrays

A dilution series of purified HCMV virions were incubated on two different custom glycoarrays (Tables 1 and 2, Z-Biotech) using established protocols [36] and the arrays were analyzed to assess specific virus binding. Briefly, 10⁵ to 10⁸ pfu/ml of purified virions were incubated for an hour on glycoarrays containing six replicates of each glycosaminoglycan. After incubation, staining with primary antibody (mouse anti gB (clone 2F12, Virusys Inc, Taneytown, MD) was done at 100 μg/ml and secondary antibody (Goat anti mouse IgG AlexaFlour555) was done at 1μg/ml. Maximum strength fluorescent signal was obtained for 10⁸ pfu/ml concentration of the virus, therefore, only this concentration is represented in the final data obtained for plotting the graphs.

Table 1. A custom designed glycoarray containing hyaluronic acid, heparin, chondroitin sulfate and dermatan sulfate species.

The structure, molecular weight, number of sugar residues and sulfate groups per disaccharide for each glycosaminoglycan are listed.

ID	Name	Structure and Molecular Weight	MW^*	No. of Sugar Residues	Sulfate Groups per Disaccharide
GAG1	Hyaluronic Acid dp10 (HA10)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₄ GlcNAc	1,950	10	0
GAG2	Hyaluronic Acid dp12 (HA12)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₅ GlcNAc	2,350	12	0
GAG3	Hyaluronic Acid dp14 (HA14)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₆ GlcNAc	2,700	14	0
GAG4	Hyaluronic Acid dp16 (HA16)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₇ GlcNAc	3,150	16	0
GAG5	Hyaluronic Acid dp18 (HA18)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₈ GlcNAc	3,650	18	0
GAG6	Hyaluronic Acid dp20 (HA20)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]₉ GlcNAc	3,900	20	0
GAG7	Hyaluronic Acid Polymer (HA93)	ΔHexAβ1,3 [GlcNAcβ1,4 GlcAβ1,3]_n GlcNAc	93,000	462	0
GAG8	Heparin dp10 (H10)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₄	3,000	10	3
GAG9	Heparin dp12 (H12)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₅	3,550	12	3
GAG10	Heparin dp14 (H14)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₆	4,100	14	3
GAG11	Heparin dp16 (H16)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₇	4,650	16	3
GAG12	Heparin dp18 (H18)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₈	5,200	18	3
GAG13	Heparin dp20 (H20)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₉	5,750	20	3
GAG14	Heparin dp22 (H22)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₁₀	6,300	22	3
GAG15	Heparin dp24 (H24)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₁₁	6,850	24	3
GAG16	Heparin dp30 (H30)	ΔHexA,2S—GlcNS,6S-(IdoA,2S-GlcNS,6S)₁₄	9,000	30	3
GAG17	Chondroitin Sulfate AC dp10 (CS10)	ΔUA—(GalNAc,6S or 4S—GlcA)₄—GalNAc,6S or 4S	2,480	10	1
GAG18	Chondroitin Sulfate AC dp12 (CS12)	ΔUA—(GalNAc,6S or 4S—GlcA)₅—GalNAc,6S or 4S	2,976	12	1
GAG19	Chondroitin Sulfate AC dp14 (CS14)	ΔUA—(GalNAc,6S or 4S—GlcA)₆—GalNAc,6S or 4S	3,472	14	1
GAG20	Chondroitin Sulfate AC dp16 (CS16)	ΔUA—(GalNAc,6S or 4S—GlcA)₇—GalNAc,6S or 4S	3,968	16	1
GAG21	Chondroitin Sulfate AC dp18 (CSD18)	ΔUA—(GalNAc,6S or 4S—GlcA)₈—GalNAc,6S or 4S	4,464	18	1
GAG22	Chondroitin Sulfate AC dp20 (CSD20)	ΔUA—(GalNAc,6S or 4S—GlcA)₉—GalNAc,6S or 4S	4,960	20	1
GAG23	Chondroitin Sulfate D dp10 (CSD10)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₄—GalNAc,6S	2,480	10	1 or 2
GAG24	Chondroitin Sulfate D dp12 (CSD12)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₅—GalNAc,6S	2,976	12	1 or 2
GAG25	Chondroitin Sulfate D dp14 (CSD14)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₆—GalNAc,6S	3,472	14	1 or 2
GAG26	Chondroitin Sulfate D dp16 (CSD16)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₇—GalNAc,6S	3,968	16	1 or 2
GAG27	Chondroitin Sulfate D dp18 (CSD18)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₈—GalNAc,6S	4,464	18	1 or 2
GAG28	Chondroitin Sulfate D dp20 (CSD20)	ΔUA—(GalNAc,6S or 4S—GlcA +/- 2S)₉—GalNAc,6S	4,960	20	1 or 2
GAG29	Dermatan Sulfate dp10 (DS10)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₄	2,480	10	1
GAG30	Dermatan Sulfate dp12 (DS12)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₅	2,976	12	1
GAG31	Dermatan Sulfate dp14 (DS14)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₆	3,472	14	1
GAG32	Dermatan Sulfate dp16 (DS16)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₇	3,968	16	1
GAG33	Dermatan Sulfate dp18 (DS18)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₈	4,464	18	1
GAG34	Dermatan Sulfate dp20 (DS20)	ΔHexA—GalNAc,4S—(IdoA—GalNAc,4S)₉	4,960	20	1

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*MW: Molecular weight (Dalton)

Table 2. A custom designed glycoarray containing different heparan sulfate species.

The structure, molecular weight, number of sugar residues and sulfate groups per disaccharide for each glycosaminoglycan are listed.

ID	Structure	MW^*	No. of Sugar Residues	Sulfate Groups per Disaccharides
HS001	GlcNAcα1-4GlcAβ1-4GlcNAcα1-4-GlcA	1000	4	0
HS002	GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcA	1,176	5	0
HS003	GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcA	1,379	6	0
HS004	GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcA	1,555	7	0
HS005	GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcA	1,758	8	0
HS006	GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAcα1-4GlcA	1,934	9	0
HS007	GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	1,076	4	1
HS008	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	1,252	5	0.8
HS009	GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	1,493	6	1
HS010	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	1,669	7	0.9
HS011	GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	1,910	8	1
HS012	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcA	2,087	9	0.9
HS013	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNS6Sα1-4GlcA	2,166	9	1.1
HS014	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcA	2,246	9	1.3
HS015	GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcA	2,327	9	1.6
HS016	GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcA	2,406	9	1.8
HS017	GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ1-4GlcNSα1-4IdoA2Sβ1-4GlcNSα1-4GlcA	1,990	8	1.3
HS018	GlcNSα1-4GlcAβ1-4GlcNSα1-4IdoA2Sβ1-4GlcNSα1-4IdoA2Sβ1-4GlcNSα1-4GlcA	2,070	8	1.5
HS019	GlcNAcα1-4GlcAβ1-4GlcNSα1-4IdoA2Sβ1-4GlcNSα1-4IdoA2Sβ1-4GlcNSα1-4GlcA	2,432	8	1.3
HS020	GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4GlcA	2,310	8	2.3
HS021	GlcNS6Sα1-4GlcAβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4GlcA	2,389	8	2.5
HS022	GlcNAc6Sα1-4GlcAβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4GlcA	2,353	8	2.3
HS023	GlcNS6Sα1-4GlcAβ1-4GlcNS3S6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4GlcA	1,893	6	2.7
HS024	GlcNAc6Sα1-4GlcAβ1-4GlcNS3S6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4IdoA2Sβ1-4GlcNS6Sα1-4GlcA	2,433	8	2.5

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*MW: Molecular weight (Dalton)

Protein expression and purification

Merlin strain HCMV gB lacking the transmembrane region [37] and codon-optimized for expression in human cells, with an optimal Kozak sequence immediately 5’ to the gene, was synthesized by Twist Biosciences (San Francisco, CA) and subcloned into pTwist CMV BetaGlobin WPRE Neoeukaryotic expression vector using the NotI and ClaI restriction sites. Purified plasmid was transfected into Expi293 cells (ThermoFisher). Culture supernatant was harvested seven days later and passed over a His Trap HP column (Cytiva) and gB protein was eluted with 450 mM imidazole. Imidazole was removed via buffer exchange using a centrifugal filter device with a 50,000 dalton cutoff (Pall Corp.). Protein purification was verified by SDS-PAGE and protein concentration was measured by BCA (ThermoScientific) (S5 Fig).

Preparation of heparin coated Surface Plasmon Resonance (SPR) sensor chip

gB protein purification and characterization of heparin analogs have been described above. Porcine intestinal heparin (15 kDa) was purchased from Celsus Laboratories (Cincinnati, OH) and N-desulfated heparin (15 kDa) and 6-O-desulfated heparin (15 kDa) were from Galen Lab Supplies (North Haven, CT). Sensor SA chips were from Cytiva Life Sciences (Uppsala, Sweden). SPR measurements were performed on a Biacore T200 operated using Biacore T200 control and T200 Evaluation software (version 3.2). Biotinylated heparin was prepared by conjugating its reducing end to amine-PEG3-Biotin (Pierce, Rockford, IL). In brief, heparin (2 mg) and amine-PEG3-Biotin (2 mg, Pierce, Rockford, IL) were dissolved in 200 μl H₂O, 10 mg NaCNBH₃ was added. The reaction mixture was heated at 70°C for 24 h, after that a further 10 mg NaCNBH₃ was added and the reaction was heated at 70°C for another 24 h. After cooling to room temperature, the mixture was desalted with the spin column (3,000 MWCO). Biotinylated heparin was collected, freeze-dried and used for SA chip preparation. The biotinylated heparin was immobilized to streptavidin (SA) chip based on the manufacturer’s protocol. The successful immobilization of heparin was confirmed by the observation of ~200 resonance unit (RU) increase on the sensor chip. The control flow cell (FC1) was prepared by 1 min injection with saturated biotin.

Kinetic measurement of interaction between heparin and gB protein using Biacore

The gB protein was diluted in HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). Different dilutions of protein samples were injected at a flow rate of 30 μL/min. At the end of the sample injection, the same buffer was flowed over the sensor surface to facilitate dissociation. After a 3 min dissociation time, the sensor surface was regenerated by injecting with 30 μL of 2M NaCl to get fully regenerated surface. The response was monitored as a function of time (sensorgram) at 25°C.

Solution competition study between heparin on chip surface and heparin analogs in solution using SPR

gB protein (250 nM) mixed with heparin (1000 nM) or heparin analogs (1000 nM) in HBS-EP+ buffer were injected over heparin chip at a flow rate of 30 μL/min, respectively. After each run, the dissociation and the regeneration were performed as described above. For each set of competition experiments on SPR, a control experiment (only protein without any heparin) was performed to make sure the surface was completely regenerated and that the results obtained between runs were comparable.

Statistics

Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test sample with control, and corrected using Dunnett’s post hoc test. In some cases, pairwise comparisons were made using Student’s t-test. Differences were considered significant if p<0.05. All statistical tests were done in Prism (Prism version 8.0, GraphPad Software, San Diego, California USA, www.graphpad.com) Standard error of mean or standard deviation was plotted as error bars. An asterisk (*) indicates significant differences compared to mock or wild-type.

Results

Purified HCMV extracellular virions preferentially bind to sulfated glycosaminoglycans with increased degree of polymerization

First, we sought to establish the category of GAG that preferentially binds to purified HCMV virions. HCMV extracellular virions were purified as described above and incubated with custom glycoarrays containing increasing molecular weight species of hyaluronic acid, heparin, chondroitin sulfate, and dermatan sulfate (Table 1). HCMV binding to non-sulfated hyaluronic acid (GAG1-GAG7) was negligent but significant binding to all heparin species was detected with a trend of increased binding to heparins as their dp increased (Fig 1A). HCMV also bound to large size chondroitin sulfate D (GAG28, dp20), and dermatan sulfate oligosaccharides (GAG32-GAG34, dp16-dp20) but not to chondroitin sulfate AC (GAG17-GAG22). It is important to note that while the chondroitin sulfate A (CS-A) is sulfated at C4 of the GalNAc, and the chondroitin sulfate C (CS-C) is sulfated at the C6 of the GalNAc only, the chondroitin sulfate D is sulfated at C2 of the glucuronic acid as well as the C6 of the GalNAc sugar and hence has double the amount of sulfation compared to CS-A and CS-C. Dermatan sulfate (DS), formerly referred to as CS-B, is formed from the polymer backbone of chondroitin sulfate by the action of chondroitin-glucuronate C5 epimerase, which epimerizes individual d-glucuronic acid residues to L-iduronic acid. The binding affinity to DS was also size-dependent increasing from GAG32-GAG34 (dp16-dp20). Heparin (dp30) was the best HCMV binder in this assay. The positive and negative controls worked as expected.

Fig 1 — **(A)** Binding of purified extracellular CMV virions on a custom designed glycosaminoglycan glycoarray. Relative fluorescence units (RFU), which are directly proportional to the amount of virus binding, are plotted on the Y-axis in the graph. Ligand descriptions and chain structures are provided in Table 1. Six replicates for each GAG were used in the assay. NC1: Negative control 1 (print buffer), NC2: (Biotinylated Glycan), PC1: positive control; human IgG (0.1 mg/ml), PC2: mouse IgG (0.1 mg/ml), PC3: rabbit IgG (0.1 mg/ml), dp: degree of polymerization, triangles at the bottom of the graph represent an increasing degree of polymerization of GAGs from the left to the right. **(B)** Binding of purified extracellular CMV on a custom designed heparan sulfate glycoarray. Relative fluorescence units (RFU) are plotted on the Y-axis in the graph. Ligand descriptions and chain structures are provided in Table 2. Six replicates for each ligand were used. NC1: negative control 1 (print buffer), NC2: (biotinylated glycan), NC3: human IgG (0.1 mg/ml), PC1: mouse IgG (0.1 mg/ml), PC2: rabbit IgG (0.1 mg/ml). Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001). Standard deviation was plotted as error bars.

On a second HS specific array (Table 2), HCMV showed strong binding to the HS with longer monosaccharide chains (HS007 to HS024) and minimal binding to unsulfated glycans (HS001-HS006) (Fig 1B). The maximum binding was observed for HS014, HS015 and HS016, which are all 6-O-S 9-mers with moderate amount of sulfation (1.3–1.8 sulfate group per disaccharide). Also, significant amount of binding was observed for 2-O-S (HS17-HS19), 6-O-S/2-O-S (HS20-22) and 2-O-S/6-O-S/3-O-S (HS23-24) HS that had high amount of sulfation (1.3–2.7 sulfate group per disaccharide) and 6–8 disaccharides per chain. Overall the data from these experiments indicate that the dp of HS as well as sulfation is important for HCMV binding.

The degree of polymerization of GAG chains impacts CMV infectivity

Glycosaminoglycans of different dp were fractionated from enoxaparin (a low molecular weight heparin). All of these GAGs are based on a HS backbone and differ in either dp or degree/place of sulfation or both (Fig 2 and S1 Fig). These GAGs, along with heparin and Arixtra (fondaparinux sodium), were first screened in a GFP-based preliminary virus focus reduction assay using GFP tagged HCMV (Towne strain). The viral GFP expression was most efficiently reduced by heparin salt (PIHSS; Heparin sodium salt from porcine intestinal mucosa) whereas Arixtra, 6-O-desulfated Arixtra and enoxaparin had little to no impact on GFP expression (Fig 2). In general, enoxaparin derived GAGs with higher dp were more efficient in reducing viral GFP compared to low dp derivatives. To follow up on this primary GFP based screening, we performed viral titer assay using HCMV (Towne strain) that measures total virus yields at 5 days post-infection. Most reduction in viral titers was observed for heparin (PIHSS) followed by enoxaparin derivative with >20 dp (Fig 3A). Plotting of viral titer reduction as a function of dp revealed a general trend where higher dp derivatives lead to higher reduction in viral titers (Fig 3B). Thus, this experiment indicated that longer HS chains are more efficient at reducing HCMV titers in cells. To investigate whether this inhibitory effect was due to an increase in the number of HCMV binding sites per chain of longer chain GAGs towards virus particles, the experiments were repeated at 0.05 g/L concentrations of GAGs instead of the previously used molar equivalent concentrations (Fig 3C). As micromolar concentration (10 μM) of GAGs is based on number of molecules provided, GAGs consisting of longer chain will have more potential binding sites for virus than those of shorter chains. The other concentration (0.05 g/L) is based on weight; thus this concentration normalizes the number of potential virus binding sites for GAGs consisting of both long and short chains. Interestingly, similar trend of inhibitory results leaning towards efficacy of higher dp against HCMV infection were obtained at 0.05 g/L indicating that this effect is not merely due to a higher number of potential independent binding sites in the longer GAG chains and instead involves a difference in the molecular interaction between HCMV and the longer GAG chains. A line graph for each concentration of GAGs was generated that demonstrates the relationship of viral titer and degree of polymerization (Fig 3D). Although GAG treatment is not known to induce cell death, to rule out that these effects on virus titers could be attributed to the health of cells, we performed cell viability assays in both uninfected and infected settings. Cell viability was not affected at the treated concentrations of any of our test GAGs (S4A Fig). Moreover, heparin (PIHSS) and some enoxaparin derivatives (dp 12 or greater) appeared to protect cells from death that was evident in mock-treated controls (S4B Fig). These results corroborate the results of our glycoarray experiments that showed that GAG with higher dp have higher CMV binding compared to GAG with lower dp (Fig 1A).

Fig 2 — Primary human foreskin fibroblasts (HFF) grown in 96 well plate were pretreated for one hour with 10 μM of 1) 6-O-desulfated Arixtra, 2) Unmodified Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) *dp2*, 6) *dp4*, 7) *dp6*, 8) *dp8*, 9) *dp10*, 10) *dp12*, 11) *dp14*, 12) *dp16*, 13) *dp18*, 14) dp20 15) > *dp20* or control (dH₂O). Cells were infected with GFP tagged HCMV (Towne strain) virus at an MOI of 3.0 in the presence of the test glycosaminoglycans, which were maintained in the cell culture medium. At 5 days post-infection, cells were fixed and number of foci (GFP) was counted in triplicate well for each sample under an epifluorescent microscope. An average of >200 foci were present in mock-treated infected cells. Percent of viral GFP was calculated compared to virus only infected control (100% GFP expression). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001).

Fig 3 — **(A)** Primary human foreskin fibroblasts (HFF) were pretreated for one hour with 10 μM of 1) 6-O-desulfated Arixtra, 2) Regular Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) *dp2*, 6) *dp4*, 7) *dp6*, 8) *dp8*, 9) *dp10*, 10) *dp12*, 11) *dp14*, 12) *dp16*, 13) *dp18*, 14) *dp20* 15) > *dp20* or control (dH₂O). Cells were infected with HCMV (Towne strain) virus at an MOI of 3.0 in the presence of test glycosaminoglycans. Cells and media were harvested at 5 days post-infection and titered for HCMV plaque forming units (pfu) on fresh fibroblasts in tissue culture dishes. Individual samples (3 replicates each) were quantified and displayed as total pfu/ml on Y-axis. **(B)** Virus titer is plotted (Y-axis) against degree of polymerization (X-axis). Data points ahead of the broken line is for a mixture of GAGs (dp>20). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. **(C)** Primary human foreskin fibroblasts (HFF) were pretreated for one hour with 0.05 g/L of 1) 6-O-desulfated Arixtra, 2) Regular Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) *dp2*, 6) *dp4*, 7) *dp6*, 8) *dp8*, 9) *dp10*, 10) *dp12*, 11) *dp14*, 12) *dp16*, 13) *dp18*, 14) *dp20* 15) > *dp20* or control (dH₂O). Cells were infected with HCMV (Towne strain) virus at an MOI of 3.0 in the presence of test glycosaminoglycans. Cells and media were harvested at 5 days post-infection and titered for HCMV plaque forming units (pfu) on fresh fibroblasts in tissue culture dishes. Individual samples (3 replicates each) were quantified and displayed as total pfu/ml on Y-axis. **(D)** Virus titer is plotted (Y-axis) against degree of polymerization (X-axis). Data points ahead of the broken line is for a mixture of GAGs (dp>20). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001).

Cell lines defective in expression of specific sulfation enzymes have reduced CMV titers and reduced virus entry

Due to species specificity of HCMV, animal models are frequently used to study CMV pathogenesis [38,39]. Studies of murine CMV (MCMV) infections of mice have served a major role as a model of CMV biology and pathogenesis [40]. Lung endothelial cell lines from adult mice were mutated for specific sulfotransferase enzymes by a conditional Cre-LoxP or a CRISPR-Cas9 based gene editing system [34,41,42]. Since previous studies showed that 3-O-S HS is important for HCMV entry in human iris stromal cells [43], we analyzed virus replication in Hs3st1 and Hs3st4 (Glucosaminyl 3-O-sulfotransferase 1 and 4, respectively) knockout cell lines as well as the Hs3st1/4 double knockout cell line. At high (5.0) as well as low (0.01) multiplicity of infection (MOI), MCMV growth was significantly reduced in the single Hs3st1 and Hs3st4 knockouts as well as in the double Hs3st1/4 knockouts, indicating that 3-O-sulfation of HS is important for HCMV infection (Fig 4). Further, we probed whether virus entry is impacted in the cells knocked out for different combinations of sulfotransferases. Expression of viral immediate early protein (IE1) has been used as a surrogate for virus entry since it’s one of the earliest events after a successful virus entry [44–46]. Results of an immunoblot probing for IE1 show that virus entry is significantly reduced in Hs3st1^-/-, Hs3st4^-/-, Hs3st1/4-double-knockout, Hs6st1^-/-, Hs6st2^-/-, and Hs6st1/2 double-knockout cells, compared to the wild-type cells (Fig 5A and 5B).

Fig 4 — Cells were grown to 90% confluency and infected with wild-type MCMV (strain K181) at low (0.01, (**A), (B**) and high (3.0) (**C), (D**) MOI. Cells and the medium were harvested at 3- and 5-days post-infection, sonicated to release the virus and diluted for plating on to wild-type MEF in tissue culture dishes in order to enumerate total MCMV pfu/ml. Results are representative of three independent replicates. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.05). Standard error of mean was plotted as error bars. An asterisk (*) indicates significant inhibition compared to wild-type. Hs3st1 and Hs3st4: Glucosaminyl 3-O-sulfotransferase 1 and 4, respectively. WT: wild type; KO: knockout.

Fig 5 — **(A)** Cells were grown to 90% confluency and infected with wild-type MCMV (strain K181) at an MOI of 3.0. Cells were harvested at 2 hours post-infection, and whole cell lysates were loaded on a polyacrylamide gel for blotting. The blots were probed with anti IE1 antibody. Beta-actin was used as a loading control. **(B)** Bands from two independent experiments were quantified by densitometry and means were plotted. Standard error of mean was plotted as error bars. Hs6st1 and Hs6st4: Glucosaminyl 6-O-sulfotransferase 1 and 4, respectively. Hs3st1 and Hs3st4: Glucosaminyl 3-O-sulfotransferase 1 and 4, respectively. WT: wild type; dko: double knockout. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test sample with control, and corrected using Dunnett’s post hoc test. Differences were considered significant (*) if p<0.05.

Kinetics measurement of gB-heparin interactions using SPR

HCMV glycoprotein B (gB) was expressed in mammalian cells, purified and used in GAG binding assays utilizing SPR. Sensorgrams of gB-heparin interaction are shown in Fig 6A. Concentrations of gB used were 1000, 500, 250, 125, and 63 nM, respectively from top to bottom. The black curves are the fitting curves using T200 Evaluation software (version 3.2). The SPR results showed the kinetic of gB-heparin interaction: association rate constant: ka = 9.8× 10³ (±68) (1/MS), dissociation rate constant: kd = 4.2×10⁻⁴ (±1.7 ×10⁻⁶) (1/S); and binding equilibrium dissociation constant (K_D = kd/ka): K_D = 4.3×10⁻⁸ (M).

Fig 6 — **(A)** SPR sensorgrams of gB-heparin interaction. Concentrations of gB (from top to bottom): 1000, 500, 250, 125, and 63 nM, respectively. The black curves are the fitting curves generated using the T200 Evaluation software (version 3.2). **(B)** Sensorgrams of solution heparin or analogs /surface heparin competition. gB concentration was 250 nM, and concentrations of heparin/analogs in solution were 1000 nM. **(C)** Bar graphs (based on triplicate experiments with standard deviation) of normalized gB binding preference to surface heparin by competing with heparin or heparin analogs in solution. ES: Enoxaparin Sodium; ES-NS: Enoxaparin Sodium N-sulfated; ES-6 DeS: Enoxaparin Sodium 6-O-desulfated; Hep: Heparin.

Solution competition study on the interaction between heparin (on surface) with gB added with heparin or heparin analogs in solution

To examine the effect of heparin structure on heparin-gB interaction, solution/surface competition experiments were performed by SPR. Heparin (1000 nM) or heparin analogs (1000 nM) in HBS-EP+ buffer were pre-mixed with gB were injected. The results show (Fig 6B and 6C) the heparin in concentration of 1000 nM completely inhibited the gB binding to surface heparin. The inhibition was greatly reduced with desulfation of heparin.

Discussion

In this study, we utilized multiple approaches, including glycoarray binding analysis, HS mimics, HS mutant cell lines, and gB-heparin binding to demonstrate that specifically sulfated HS with higher degree of polymerization affect CMV infection and binding. The results significantly advance the age-old knowledge of HS binding to herpesviruses by illustrating the importance of HS structural modifications in CMV binding and infection. We first screened several sulfated or unsulfated GAGs with complex sugar structure to investigate which GAGs are more efficient at binding to HCMV virions. This glycoarray analysis indicated that HCMV bound heparins with strong affinity and showed increased affinity for longer chain length heparins (Fig 1A). Binding of CS was limited to the highest dp of only CS-D, a CS chain that is primarily sulfated at the C-2 position of GlcA and the C-4 position of GalNAc. CS-D has a much higher sulfate density compared to CS-A/CS-C (which are primarily only monosulfated at the GalNAc at the C-4 and C-6 positions, respectively), and the 2-O-sulfation of the GlcA is similar to the common 2-O-sulfation of IdoA found in heparin. Dermatan sulfate also only bound significantly in the highest dp tested. While DS has a sulfate density similar to CS-A/CS-C (one sulfo group per disaccharide, found at the GalNAc), it also bears similarity to heparin in that it has IdoA in its disaccharide repeat as most commonly found in heparin, unlike CS which has GlcA (differing by epimerization at the C-5 position). We would hypothesize that it is these heparin-like qualities that causes CS-D and DS to bind at high dp.

To further investigate this binding, we utilized another glycoarray consisting of HS of varied polymerization and sulfation levels. The results from this glycoarray indicated that HCMV binds strongly with HS having both longer monosaccharide chain and a moderate level of sulfation (Fig 1B). Thus, sulfated HS with more complex branches and sulfation patterns preferentially bind to HCMV. Next, we fractioned HS by length (dp 2–20) from enoxaparin and tested their ability to inhibit HCMV growth in cell culture by competing with HCMV binding. The GFP tagged HCMV was used and the number of GFP+ foci was quantified in the presence of increasing HS chain length. Amounts of viral GFP was more effectively reduced when cells were pretreated and maintained with GAGs having a higher dp (Fig 2). This assay served as a surrogate for a virus entry assay since the GFP is independently expressed from an early promoter in the virus genome [47]. For a deeper understanding of this reduction, we performed a similar experiment where HCMV Towne strain was used and viral load was quantified at 5 days post-infection (Fig 3). Significant reduction in virus titers was observed in samples treated with higher dp of GAG but not with lower dp corroborating the results from glycoarray experiments that chain length of GAG is an important factor in determining HCMV binding. Also, this effect was not due to a simple increase in the number of potential HCMV binding sites per mole of GAG, as evidenced by similar trend of inhibition obtained when treating cells with equivalent μM or g/L concentrations of GAGs. Treatment of cells with these GAGs did not affect cell viability for the duration of treatment (S4A Fig) confirming that the observed reduction in virus titer was not due to the cell death. Moreover, cells pretreated and maintained with GAGs of longer dp resisted infection induced cell death at late time post-infection (S4B Fig). We also tested the impact of specific HS sulfation mutants on MCMV infection. As 3-O-sulfation has been reported to be critical for herpesvirus entry [43,48], we tested MCMV growth in Hs3st1, Hs3st4 and dual Hs3st1/4 knockout cells. For both high and low MOI, virus titer was significantly reduced in Hs3st1, Hs3st4 and dual Hs3st1/4 knockout cells (Fig 4). Although the differences were statistically significant, we did not see a robust inhibition of virus titers in these assays compared to the wild-type. To directly assess the impact of HS sulfation on virus entry, we used several mutant cell lines deficient in HS sulfation enzymes. All of these cells were defective in virus entry as assessed by IE1 protein expression (Fig 5). The GAG experiments used a fibroblast cell culture system and a fibroblast tropic strain of HCMV (Towne), whereas MCMV experiments used lung epithelial cells, thus providing the experimental data from multiple cell types and two different viruses. While the GFP based assays provide a surrogate for virus entry assays, the real impact of virus entry inhibitors would be a reduction in viral titers at the end of infection since an entry inhibitor that only delays virus entry would be of little translational value. Thus virus yield and titers were used a measure of effectiveness of GAG inhibitors.

The enzymatic modification of HS chains is known to generate unique binding sites for viral ligands. For example, 3-O-sulfation modification in HS chain generates fusion receptor for HSV glycoprotein D (gD) promoting viral entry and spread [49]. The 3-O-S HS is a product of enzymatic modification at C3 position of glucosamine residue, which is relatively rare in comparison to other HS modifications. Expression of Hs3st can make normally resistant Chinese hamster ovary (CHO-K1) cells susceptible to HSV-1 infection [50]. Studies in clinically relevant primary human corneal fibroblasts have also shown 3-O-S HS as a primary attachment receptor for HSV entry [48]. Interestingly, both HSV-1 and HSV-2 use HS as an attachment receptor but HSV-1 binds to distinct modification sites on HS that HSV-2 is unable to, which could explain some of the differences in cell tropism exhibited by these two viruses [51]. For example, while N-sulfation and carboxyl groups are required for both HSV-1 and HSV-2 binding, only HSV-1 is able to bind the specific modification sites generated by 2-O-, 6-O-, and 3-O-sulfations [52]. The O-desulfated heparins have little or no inhibitory effect on HSV-1 infection but inhibit HSV-2 infection. This susceptibility to O-desulfated heparins can be transferred to HSV-1 by recombinant transfer of the gene for glycoprotein C (gC-2) from HSV-2 [52]. We reported earlier that 3-O-S HS are important for HCMV entry in human iris stromal (HIS) cells [43]. The expression of Hs3st in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or treatment with anti-3-O-S HS (G2) peptide significantly reduced HCMV plaques/foci formation. In addition, co-culture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-O-S HS significantly enhanced cell fusion. A similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) co-cultured with 3-O-S HS cells. These results highlight the role of 3-O-S HS during HCMV entry.

Herpesvirus glycoprotein B is one of the most conserved glycoprotein across the herpesvirus family [53]. Herpes Simplex Virus -1 (HSV-1) gB and gC have been shown to be responsible for HS binding on cell surface [8,54]. However, HCMV is not known to possess a sequence homolog of gC. Thus, we expressed and purified HCMV gB and used it in a GAG binding assay using SPR. This gB showed a very strong binding to heparin. Upon competition with different heparin analogs, it was found that only heparin itself and a 6-desulfated form of heparin significantly competed with gB-bound heparin. Enoxaparin and its desulfated homolog were unable to compete with heparin. These results confirm the results in our GAG screening assay where the sulfated forms of heparin were most effective in blocking virus infection.

Owing to their inherent structural features, certain sulfated glycans can exert therapeutic effects against infections caused by pathogenic microorganisms. A study by Pomin et al., showed that administering sulfated glycans can disrupt the pathogen protein-host glycosaminoglycan (GAG) complex formation causing impairment of microbial binding onto host cells [55]. Similarly, sulfated GAG, glycosphingolipids and lectins have been shown to inhibit DENV entry [56]. Heparan sulfate mimics, such as suramin, pentosan polysulfate, and PI-88, SPGG [57,58] have been reported to be effective against multiple viruses including herpesviruses [4,59,60]. The inhibitory activity of HS mimics, including these compounds, is believed to be due to their association with GAG binding sites of the putative receptor-binding domain on the viral protein [4,61]. Thus, HS mimics can inhibit virus adsorption and entry.

Overall, the data from these studies indicate that dp of GAGs as well as specific sulfation patterns govern CMV infection of cells. These studies show the promise of highly polymerized sulfated-HS as effective anti-CMV agents. Future studies will be aimed at confirming the CMV glycoproteins that specifically bind to HS on cell surface and their possible structural illustrations.

Supporting information

S1 Fig. Bio-Gel P10 size exclusion column chromatogram of enoxaparin separation.

Fractions were collected and UV readings at 232 nm were taken for each fraction to reconstruct the chromatogram. Samples were pooled to obtain the oligosaccharide fractions of the desired size.

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S2 Fig. ESI-MS of 6-O-desulfated Arixtra.

The most abundant MS masses were consistent with the loss of the three 6-O-sulfates from Arixtra.

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S3 Fig. MS/MS analysis of the -4 charge state of Arixtra-3SO₃.

Glycosidic bond cleavages isolate desulfation to one desulfation event in the two non-reducing end residues; one desulfation event in the two reducing end residues, and one desulfation event in the central GlcNS. This pattern is consistent with 6-O-desulfation.

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S4 Fig. Effect of GAG treatment on cell viability of HFF cells.

Primary HFF were pretreated for one hour with 10 μM of 1) 6-O-desulfated Arixtra, 2) Regular Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) dp2, 6) dp4, 7) dp6, 8) dp8, 9) dp10, 10) dp12, 11) dp14, 12) dp16, 13) dp18, 14) dp20, 15) > dp20 or control (dH₂O). Cells were either mock infected (A) or infected with HCMV (Towne strain) virus at an MOI of 3.0 (B) in the presence of test glycosaminoglycans. Cells were harvested at 5 days post-infection and cell viability was assessed using Trypan Blue exclusion assay. Results are representative of three independent replicates. Standard error of mean was plotted as error bars.

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S5 Fig. Expression and analysis of HCMV glycoprotein B (gB).

HCMV gB was expressed and purified for GAG binding assays as described in Materials and Methods. Protein purification was verified by SDS-PAGE followed by staining with Coomassie Brilliant Blue. (A) Coomassie protein gel of purified CMV gB as well as flow thorough and washes. (B) CMV gB protein gel lane analysis was performed by using Bio-Rad Image Lab Software. Coomassie purity of gB was determined to be 100% and molecular weight was 107.59 kD. Lane 1—protein ladder (Bio-Rad Precision Plus Protein Dual Color Standards), Lane 2—CMV gB column flow through, Lane 3—1^st wash, Lane 4—2^nd wash, Lane 5—blank, Lane 6—CMV gB.

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Acknowledgments

We would like to thank Jian Zhang and Jessica Kelly at Z Biotech, LLC (Aurora, Colorado, USA) for help with glycoarray experimental design and data analysis.

Data Availability

All data are in the manuscript and/or Supporting information files.

Funding Statement

The research was supported by NASA (Award #80NSSC19K1603, PI: RT) and NIH (Award 5U01CA225784, PI: LW). QL, JSS and LW acknowledge funding from the National Institute of General Medical Sciences through the Research Resource for Integrated Glycotechnology (P41GM103390). RL and FZ were supported by NIH for T200 Biacore instrument grant (S10OD0285232). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1009803.r001

Decision Letter 0

Blossom Damania, Eain A Murphy

24 Apr 2021

Dear Dr. Tandon,

Thank you very much for submitting your manuscript "The Degree of Polymerization and Sulfation Patterns in Heparan Sulfate are Critical Determinants of Cytomegalovirus Entry into Host Cells" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers.

We sent the manuscript to three experts with expertise in herpesviral attachment and entry. All reviewers were in agreement that while very interesting, there were major concerns about the work and some of the data are self-contradictory. Additionally, some of the reviewers were not convinced about the novely of the experiments. The reviewers' comments are listed below and we hope you will find them helpful. If you are able to answer these concerns, we will be happy to consider a revised manuscript. Please note that the revised manuscript will be sent back to the reviewers for re-evaluation.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Eain Murphy

Associate Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript uses novel glyco arrays, peptides, and sulfation enzyme knockout lines in an attempt to map whether the degree of sugar polymerization and/or sulfation is important for MCMV or HCMV entry.

Reviewer #2: The authors have tried to demonstrate that structural specificities including sulfation patterns within heparin/Heparan sulfate play important roles in CMV entry and infectivity. While the area of research may be significant, many additional experiments are needed to improve novelty and reduce confusion and self contradiction.

Reviewer #3: The manuscript by Hasan et al addresses the role of specific cellular glycan modifications to infection by human cytomegalovirus. Glycan modification of mammalian cell surface proteins are complex with a tremendous variation in the lengths of the polymer chains and the chemical linkages between the different carbohydrate moieties. Many viruses use glycan modifications as receptors and the various branched structures can have major impacts on tropism; specific types of structures being characteristic of specific tissues or even mammalian species. The general view is that herpesviruses use heparin sulfate glycan modifications for initial adsorption, which then facilitates the binding to specific entry receptors (e.g., nectin for HSV). Here, Hasan and colleagues characterize the use of HS for cytomegalovirus infection, focusing on the polymer length and the sulfate linkage. They use an array approach to measure the binding of CMV to a variety of synthetic glycosaminoglycans and hone in on specific heparan sulfate characteristics for further study. Further experiments showing that soluble HS can interfere with CMV infection, and experiments using cell lines deficient in the enzymes that generate specific sulfate linkages lead the authors to conclude that the CMV bind principally to HS comprised of longer polymer chains bearing a 3-O-S sulfate linkage. Authors’ then speculate on the significance of this conclusion suggesting potential use in development of antiviral therapeutics.

A number of concerns outlined below limit enthusiasm for the study. In summary: 1) Experimental rigor is low given the lack of appropriate and rigorous statistical analyses of significances (especially in cases where the measured differences are small.) Moreover, some experimental protocols do not seem well matched to the hypotheses and this creates valid, alternative interpretations (see below for more detail). Together these concerns cast doubt on the validity of the conclusions. 2) The studies were conducted with only the “Towne” strain of HCMV. Given what is now appreciated about the genetic diversity of HCMV in vivo, including additional strains would increase the impact of the study. As a related point, there was not effort to identify what HCMV glycoprotein(s) are involved in the proposed interactions. Prior research has implicated gB and gM/gN in binding to GAGs, but in neither case has this been extensively studied. Both glycoprotein complexes exhibit some degree of genetic diversity. 3) A better discussion of the benefits and drawbacks of developing therapeutics targeting this stage of HCMV infection would increase the impact of the study. - Brent Ryckman (University of Montana)

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1) needs to have the full panel of mouse GAG modification knockout lines tested.

2) Characterization/quantification of the purity of their different fractionation experiments

3) Inhibition analysis with different tropic strains of HCMV

Reviewer #2: Fig. 1 is unnecessary. Similar or identical figures exist in many papers including at least one by the authors here (PMID: 22773448). Please just refer to the published paper(s).

Figs.2/3, this data will be more meaningful (informative) if binding to gB was also measured in parallel and compared with the data shown here. This would also allow the authors to measure the binding affinity more precisely, something that has been hard to achieve in the field. Also, how do heparin structures (sulfation patterns etc.) compare with HS structures present in the glycoarray shown in Fig. 3?

Fig. 3, instead of Fig.1 the common heparin and HS structures (disaccharide or higher) that contribute to binding should be shown.

Fig. 4, the results are confusing and correlation with heparin/HS structures is not well described. Higher polymerization means higher negative charge availability to bind positive residues on gB or viral envelope, nothing specific or truly novel here. If the point is to show that heparin blocks viral growth then that point was already shown by Teresa Compton and colleagues almost three decades ago (PMID: 83847).

Figs. 5/6, argument similar to above (#4) applies here. Also, what is the new message here? Basically, multiple repetitions of results from Fig. 4. Not very impressive inhibition of infection. No statistics is performed to determine the significance.

Figs. 8/9, the results do not compare well. While the virus replicates the worst in 3OST1 KO cells (Fig. 8) it shows the best entry in the same cells (Fig. 9). Contradictory results, what is going on? Also, why does the double knockout support better replication (Fig. 8 statistical analysis suggests that results are significant)? Structural analysis of the GAGs from the double KO should be made and compared with single KOs.

Fig. 10, what is the purpose or novelty of this experiment? Similar use of anti HS peptides has been demonstrated against CMV in multiple papers by Tim Sparer’s group.

Reviewer #3: Suggested new experiments to strengthen the study.

• Some indication of that HCMV glycoprotein(s) are involved in the interactions proposed.

• I don’t understand why the experiments in Figs 4-5 were done by pre-incubating the cells with the GAG derivatives. If the hypothesis is that the virus binds to these GAG structures on the cells, then would it not make more sense to pre-incubate the virus with the GAGs to “block” the sites on the virus before you add the virus to the cells (Like using soluble receptor). This would also lessen the likelihood of effects on the cell physiology.

• Showing “dose responses” in experiments such as those in Figs 4-5 (potentially others) would also help strengthen the conclusion of preferential binding to specific GAGs.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Really arrays in Fig. 2/3 could be combined into 1 figure A and B. Figure 3 is a just a more detailed analysis of the HS array in figure 2.

Previous publications do not indicate that CS or DS affect HCMV binding. Why does this array disagree with this? Length of the GAGs or sulfation? How much do these arrays relate to what is seen on actual susceptible cells?

Fig. 5 the degree of reduction is only 1-1.5 log reduction in titers at the highest dp. Although statistically significant, it also means that ~90% of the virus is NOT dependent on the dp and or other entry mechanisms.

Fig. 7 could be moved to supplemental data as this would not be expected to cause cell death. Important but not necessary in the body of the paper.

Fig. 8 These MCMV cell lines only show a modest reduction implying multiple HS/modifications are important for infection. There are a whole panel of the these lines available from the Wang /Esko lab in order to provide a more complete picture of the important of the different sulfations on viral entry. Their paper from 2018 is pretty comprehensive as they knocked out a many of the enzymes involved in the HS/sulfation pathway. There needs to be an explanation for why the double KO does not lead to a “cumulative” (as mentioned in the discussion).

The switching and in complete analysis between HCMV and MCMV is confusing. Complete the analysis in either one or the other. Much of the previous works has been completed for HCMV so a complete analysis of MCMV entry would be more novel.

An interesting concept that could make the manuscript more complete/interesting would be the inhibition experiments with endothelial, fibroblast, macrophage-tropic viruses and how they are or are not interfered with the dp HS/sulfations.

Fig. 9 This is unconvincing and includes mutant lines not described/used in Fig. 8. There are potentially interesting cell line mutants that could provide interesting information if carried out as in Fig. 8’s set up. It would be better to use a GFP expressing virus to address entry instead of these western blots.

Fig. 10 The number of foci/well are too low. Usually (for consistency) you want closer to 50 /well. There are many different types of HS binding peptides. There were from Tiwari et al but there are others out there that block entry more generally and would be good to use as a positive/negative control. They would need to show the specificity of theses peptides on the cell types used. Why not see if they block entry on the different mouse KO lines? Would it further decrease entry/infection?

Should be using STD not SEM for figures. Also should show each data point to help in the interpretation.

Reviewer #2: (No Response)

Reviewer #3: • Line 70. Didn’t Hetzenecker 2015 show that HCMV can also use endocytosis pathways in fibroblasts?

• Fig2. Is there some kind of statistical error/significance test that can be used? The legend indicates six replicates, which implies that the values plotted are averages, is this the case? Fig 3 is the same type of analysis and has error bars, although the legend does not state what they represent. In any case, some analysis beyond standard deviation or standard error would likely be called for here.

• Materials and methods indicates that HCMV Towne strain was used. Could the authors provide more information. Is this BAC derived? Or the ATCC Towne? (which is a mix of genotypes, right? Long and short at least?). Or is this some other variant of Towne? Moreover, given the explosion of research relating to HCMV genetic diversity, how can the authors conclude that this heparin interaction is characteristic of all strains or variants of HCMV? Particularly since there is no indication of what CMV glycoproteins are binding these GAGs (assuming this conclusion is true: see other comments).

• Line 249: I understand the point of this control, but if it is a negative control, then why name it a positive control only to have to explain here?

• Fig 4. It is unclear from the results text or the legend if the GAG derivatives were present throughout the 5 day experiment. Maybe it is in the materials and methods, but it is important enough to be stated in the legend. Is this testing initial infection (entry) or also subsequent spread over 5 days? Again, some statistical analysis beyond standard error would be good, especially since the data are normalized to %. What was the dynamic range? (how many foci were counted in the control wells?

• Fig 5 seems to be the same experiment as in Fig 4, but with an alternative readout (viral titer recovered at 5 days vs # of GFP+ foci at 5 days). What is the point of the plot in 5B? Is seems to be only a subset of the exact data from 5A, just plotted as dots connected by a line rather than as columns. If you are trying to show a correlation with the degree of polymerization, there are specific correlation tests that may be appropriate. The more interesting comparison would seem to be between the two readouts; ie, why does the titer recovered at 5 days not track with the GFP readout? If the GAG derivatives are present for the entire experiment, are the progeny release to the supernatant affected? Does this effect then carry over to the plaque assay titration? Or are the cells themselves affected by the GAGs?

• Fig 7A. Vital dye exclusion is a very coarse assessment of potential effects of the GAGs on the cells. Is it possible that the physiology of the cell is altered in a way that does not “kill” the cell?

• Line 294/Fig7B. Statement in results does not seem to match data shown.

• Fig 8. Legend states that the error bars represent standard error, but there is no indication of the statistical tool used for the “asterisks” of significance. If these asterisks are only indicating which error bars do not overlap with the control, they should be removed and an appropriate test used. (Probably one-way ANOVA+Dunnetts post hoc). Some of the differences seem quite small.

• Fig 9. It would be much easier to interpret these data if the lane order in the blots matched the order of the relative density columns left to right.

• Lines 315-318. Is it really fair to say that the data in Fig 9 indicate differences in “entry?” I grant you that the notion suggested above that the GAG derivatives might affect cell physiology is a bit of the “formal caveat” (meaning it is formally possible, but maybe not all that likely). But here in fig 9, the cells have been asked to grow without the normal GAGs modification pathways. This, by definition, means their physiology is altered, no? I’m no expert on glycan modifications, but seems to me that the pathways are critical to the cell being able to move membrane proteins from the ER to the plasma membrane and then recycle them. It’s hard to imaging these cells are not physiologically abnormal. Maybe they just can’t support IE expression very well. Even if the cells are able to support IE expression normally after entry, how would you know that the entry defect is due to the lack of binding to the GAGs and not that the altered glycan processing pathways impacted the expression of specific receptors (if this was an HCMV experiment, I would suggest PDGFR-alpha, for example)?

• Line322. Is there some citation or supplemental data to support the specificity of these peptides as stated?

• Fig 10. As suggested above, is it possible that these peptides alter cell physiology? In fact, this seems likely in this experiment since they are designed to actually bind to the cells, and may well engage signaling pathways similar to the natural ligands for the GAGs as indicated by the authors in Lines 42-43 (cytokines; growth factors etc).

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PLoS Pathog. 2021 Aug 5;17(8):e1009803. doi: 10.1371/journal.ppat.1009803.r002

Author response to Decision Letter 0

13 Jul 2021

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PLoS Pathog. doi: 10.1371/journal.ppat.1009803.r003

Decision Letter 1

Blossom Damania, Eain A Murphy

15 Jul 2021

Dear Dr. Tandon,

We are pleased to inform you that your manuscript 'The Degree of Polymerization and Sulfation Patterns in Heparan Sulfate are Critical Determinants of Cytomegalovirus Entry into Host Cells' has been provisionally accepted for publication in PLOS Pathogens.

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Dear Dr. Tandon,

We, the editors, have reviewed your resubmission and have come to the conclusion to render a decision of Accept for this version as it is apparent that you have taken the original reviewers' concerns seriously, have made significant modifications to the manuscript and have vastly improved the work in doing so.

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Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1009803.r004

Acceptance letter

Blossom Damania, Eain A Murphy

3 Aug 2021

Dear Dr. Tandon,

We are delighted to inform you that your manuscript, "The Degree of Polymerization and Sulfation Patterns in Heparan Sulfate are Critical Determinants of Cytomegalovirus Entry into Host Cells," has been formally accepted for publication in PLOS Pathogens.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Bio-Gel P10 size exclusion column chromatogram of enoxaparin separation.

Fractions were collected and UV readings at 232 nm were taken for each fraction to reconstruct the chromatogram. Samples were pooled to obtain the oligosaccharide fractions of the desired size.

(TIF)

Click here for additional data file.^{(3.1MB, tif)}

S2 Fig. ESI-MS of 6-O-desulfated Arixtra.

The most abundant MS masses were consistent with the loss of the three 6-O-sulfates from Arixtra.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S3 Fig. MS/MS analysis of the -4 charge state of Arixtra-3SO₃.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S4 Fig. Effect of GAG treatment on cell viability of HFF cells.

(TIF)

Click here for additional data file.^{(4.4MB, tif)}

S5 Fig. Expression and analysis of HCMV glycoprotein B (gB).

(TIF)

Click here for additional data file.^{(6.4MB, tif)}

Attachment

Submitted filename: PLoS Pathogens Review 07092021.docx

Click here for additional data file.^{(35.4KB, docx)}

Data Availability Statement

All data are in the manuscript and/or Supporting information files.

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PERMALINK

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Dipanwita Mitra

Mohammad H Hasan

John T Bates

Michael A Bierdeman

Dallas R Ederer

Rinkuben C Parmar

Lauren A Fassero

Quntao Liang

Hong Qiu

Vaibhav Tiwari

Fuming Zhang

Robert J Linhardt

Joshua S Sharp

Lianchun Wang

Ritesh Tandon

Roles

Abstract

Author summary

Introduction

Materials and methods

Preparation of glycosaminoglycans (GAGs) oligosaccharides

Preparation of the 6-O-desulfated Arixtra with MTSTFA

LC-MS/MS analysis

Cells

Virus

Virus infection and cell viability assay

Virus titers

Immunoblots

Glycoarrays

Table 1. A custom designed glycoarray containing hyaluronic acid, heparin, chondroitin sulfate and dermatan sulfate species.

Table 2. A custom designed glycoarray containing different heparan sulfate species.

Protein expression and purification

Preparation of heparin coated Surface Plasmon Resonance (SPR) sensor chip

Kinetic measurement of interaction between heparin and gB protein using Biacore

Solution competition study between heparin on chip surface and heparin analogs in solution using SPR

Statistics

Results

Purified HCMV extracellular virions preferentially bind to sulfated glycosaminoglycans with increased degree of polymerization

Fig 1. Binding of human cytomegalovirus on glycosaminoglycan glycoarrays.

The degree of polymerization of GAG chains impacts CMV infectivity

Fig 2. Inhibition of HCMV growth by glycosaminoglycan derivatives.

Fig 3. Effect of glycosaminoglycan derivatives on HCMV growth.

Cell lines defective in expression of specific sulfation enzymes have reduced CMV titers and reduced virus entry

Fig 4. Mouse CMV replication in sulfotransferase knockout cell lines.

Fig 5. Mouse CMV entry in sulfotransferase knockout cell lines.

Kinetics measurement of gB-heparin interactions using SPR

Fig 6. gB-heparin interaction.

Solution competition study on the interaction between heparin (on surface) with gB added with heparin or heparin analogs in solution

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Blossom Damania

Eain A Murphy

Roles

Author response to Decision Letter 0

Decision Letter 1

Blossom Damania

Eain A Murphy

Roles

Acceptance letter

Blossom Damania

Eain A Murphy

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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