Antiviral activities of four marine sulfated glycans against adenovirus and human cytomegalovirus

Abstract

Broad-spectrum antivirals are more needed than ever to provide treatment options for novel emerging viruses and for viruses that lack therapeutic options or have developed resistance. A large number of viruses rely on charge-dependent non-specific interactions with heparan sulfate (HS), a highly sulfated glycosaminoglycan (GAG), for attachment to cell surfaces to initiate cell entry. As such, inhibitors targeting virion-HS interactions have potential to have broad-spectrum antiviral activity. Previous research has explored organic and inorganic small molecules, peptides, and GAG mimetics to disrupt virion-HS interactions. Here we report antiviral activities against both enveloped (the herpesvirus human cytomegalovirus) and non-enveloped (adenovirus) DNA viruses for four defined marine sulfated glycans: a sulfated galactan from the red alga Botryocladia occidentalis; a sulfated fucan from the sea urchin Lytechinus variegatus, and a sulfated fucan and a fucosylated chondroitin sulfate from the sea cucumber Isostichopus badionotus. As evidenced by gene expression, time of addition, and treatment/removal assays, all four novel glycans inhibited viral attachment and entry, most likely through interactions with virions. The sulfated fucans, which both lack anticoagulant activity, had similar antiviral profiles, suggesting that their activities are not only due to sulfation content or negative charge density but also due to other physicochemical factors such as the potential conformational shapes of these carbohydrates in solution and upon interaction with virion proteins. The structural and chemical properties of these marine sulfated glycans provide unique opportunities to explore relationships between glycan structure and their antiviral activities.

Keywords: Adenovirus; Antiviral; Broad-spectrum; Cytomegalovirus; Marine sulfated glycans; Virion attachment.

Fig. 1.. Structures and size characterization of the sulfated marine glycans.

(A-E) Structures are shown for heparin and the four sulfated marine glycans (counterions omitted for clarity). (F) The indicated sulfated glycans (10 μg) were separated by polyacrylamide gel electrophoresis and stained with toluidine blue. To the right are indicated MWs of standard compounds LMWH (7.5 kDa), UFH (15 kDa), CS-A, (40 kDa), and CS-C (60 kDa).

Fig. 2.. HCMV and Ad5 antiviral activities and cytotoxicities of marine sulfated glycans.

(A) Anti-HCMV activity (black) was measured by incubating MRC-5 fibroblast monolayers in 96-well plates with BoSG, LvSF, IbSF, IbFucCS, or heparin (HEP) for one h, then infecting with GFP-tagged HCMV BADr (100 PFU/well). (B) Anti-adenovirus activity (black) was measured by incubating ARPE-19 epithelial cell cultures as above then infecting with GFP-tagged Ad5 (100 PFU/well). After six days GFP activities in cultures were measured. Cell viability (red) was measured in replicate uninfected cultures treated for five days using the CellTiter-Glo® assay. Data are means of triplicate experiments ± standard deviations.

Fig. 3.. Marine sulfated glycans inhibit viral-encoded GFP expression when present prior to HCMV or Ad5 infection.

(A) Confluent monolayers of MRC-5 fibroblasts or ARPE-19 epithelial cells in 96-well plates were treated with medium (Ø) or with 150 μg/mL BoSG, LvSF, IbSF, IbFucCS, or heparin (HEP) either one h before or one h after infection with GFP-tagged HCMV BADr (100 PFU/well) or GFP-tagged Ad5 (100 PFU/well). Representative fluorescent micrographs were taken six days post infection. (B) Confluent monolayers of MRC-5 fibroblasts or ARPE-19 epithelial cells were treated as above either one h before, concurrent with, or 1, 3, 6, 12, or 24 h after infection with GFP-tagged HCMV BADr (100 PFU/well) or GFP-tagged Ad5 (100 PFU/well). GFP expression was quantified on day six post infection. Data are means of triplicate wells ± standard deviations.

Fig. 4.. Marine sulfated glycans inhibit expression of HCMV IE1/2 proteins and deposition of tegument protein pp65.

(A) MRC-5 fibroblast monolayers were treated for one h with medium (Ø) or 150 μg/mL heparin (HEP), BoSG, LvSF, IbSF, or IbFucCS, then infected with HCMV RC2626 (125 PFU/well). Cultures were fixed and fluorescently stained for HCMV IE1/2 proteins 48 h post infection. (B) MRC-5 fibroblast monolayers were treated as above but incubated with HCMV BADr (200 PFU/well) for one h at 4°C. Cultures were then shifted to 37°C and incubated for six h before being fixed and fluorescently stained for the HCMV tegument protein pp65.

Fig. 5.. Treatment/removal studies.

(A) Confluent monolayers of MRC-5 fibroblasts (top) or ARPE-19 epithelial cells (bottom) in 96-well plates were treated with medium (Ø) or 150 μg/mL heparin (HEP), BoSG, LvSF, IbSF, or IbFucCS for one h then cells were washed three times with medium and infected with GFP-tagged HCMV BADr (100 PFU/well) or GFP-tagged Ad5 (100 PFU/well). (B) HCMV BADr or adenovirus virions were incubated with sulfated glycans as in (A) for 1 h, then diluted 10,000-fold with culture medium to a non-inhibitory concentration (15 ng/mL). Virions with sulfated glycans were then added to MRC-5 fibroblasts (top) or ARPE-19 epithelial cells (bottom) in 96-well plates. Representative fluorescent micrographs were taken six days post infection.