Sharma P et al. (2023), Inhibition of Cytomegalovirus by Pentacta pygma...

ECB-ART-51454

Viruses 2023 Mar 28;154:. doi: 10.3390/v15040859.

Show Gene links Show Anatomy links

Inhibition of Cytomegalovirus by Pentacta pygmaea Fucosylated Chondroitin Sulfate Depends on Its Molecular Weight.

Sharma P , Dwivedi R , Ray P , Shukla J , Pomin VH , Tandon R .

???displayArticle.abstract???
Many viruses attach to host cells by first interacting with cell surface proteoglycans containing heparan sulfate (HS) glycosaminoglycan chains and then by engaging with specific receptor, resulting in virus entry. In this project, HS-virus interactions were targeted by a new fucosylated chondroitin sulfate from the sea cucumber Pentacta pygmaea (PpFucCS) in order to block human cytomegalovirus (HCMV) entry into cells. Human foreskin fibroblasts were infected with HCMV in the presence of PpFucCS and its low molecular weight (LMW) fractions and the virus yield at five days post-infection was assessed. The virus attachment and entry into the cells were visualized by labeling the purified virus particles with a self-quenching fluorophore octadecyl rhodamine B (R18). The native PpFucCS exhibited potent inhibitory activity against HCMV specifically blocking virus entry into the cell and the inhibitory activities of the LMW PpFucCS derivatives were proportional to their chain lengths. PpFucCS and the derived oligosaccharides did not exhibit any significant cytotoxicity; moreover, they protected the infected cells from virus-induced lytic cell death. In conclusion, PpFucCS inhibits the entry of HCMV into cells and the high MW of this carbohydrate is a key structural element to achieve the maximal anti-viral effect. This new marine sulfated glycan can be developed into a potential prophylactic and therapeutic antiviral agent against HCMV infection.

???displayArticle.pubmedLink??? 37112839
???displayArticle.pmcLink??? PMC10142442
???displayArticle.link??? Viruses
???displayArticle.grants??? [+]

???attribute.lit??? ???displayArticles.show???

	Figure 1. Structural representation of sulfated glycans assayed for anti-HCMV activity. (A) Unfractionated heparin (UFH) is mostly composed of repeating disaccharide units of [→4)-α-GlcN-(1→4)-α-IdoA-(1→] where GlcN is glucosamine and IdoA is iduronic acid. Sulfation occurs frequently at the N- and C6-positions of GlcN and C2 position of IdoA. (B) PpFucCS is constituted of a chondroitin sulfate backbone that alternates N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) in repeating disaccharide units of [→3)-β-GalNAc-(1→4)-β-GlcA-(1→], where the GalNAc units are primarily 4-sulfated (80%) and, to a very less degree, 4,6-disulfated (10%) or nonsulfated (10%). The GlcA units are replaced at the C3 position by three different forms of α-fucose (Fuc) branches: Fuc2,4S-(1→(40%), Fuc2,4S-(1→4)-Fuc-(1→(30%), and Fuc4S-(1→(30%); where S = SO3−.
	Figure 2. Anti-HCMV activity of PpFucCS oligosaccharides. PpFucCS oligosaccharides were assayed for their potential to inhibit the HCMV (GFP-tagged TowneBAC strain) infection in HFFs (MOI 0.1) by enumerating the cells expressing GFP. (A) Normalized values from the assay were analyzed by nonlinear regression to fit a dose–response curve using the least squares method considering each repeated measure as an individual point. The plotted curve shows the percentage of HCMV inhibition in a (log) concentration-dependent manner. Curves in the plot represent the following: PpFucCS (red), HdPpFucCS (green), Fr1 (navy), Fr2 (purple), Fr3 (blue), Fr4 (brown), and UFH (black). (B) Using the normalized values from the same assay, we calculated the percentage inhibition at the maximum concentration used (Imax) and compared with the UFH at the same concentration (50 µg/mL) by performing one-way ANOVA test with multiple comparisons by comparing the means of each test with control UFH, and corrected using a Dunnett’s post hoc test, showing significant differences among the means. The results are representative of three independent experiments. The standard error of the mean was plotted as error bars. The *, , and ns (non-significant) indicate p-value < 0.001, from 0.001 to 0.01, and ≥0.05, respectively.
	Figure 3. Inhibition of HCMV entry into the HFF cells by native PpFucCS. The primary HFF were challenged by the unlabeled (a) or R18 labeled (b–i) TowneBAC strain of HCMV at a MOI of 3.0. The HFFs were either mock treated (a–c) or pretreated for one hour using PpFucCS (50 μg/mL) (d–i). The number of fluorescent virus particles were enumerated at different time points post infection and plotted in Graphpad Prism 9. The HFFs were either mock treated and infected with the unlabeled virus at ‘60′ min (a), or mock treated and infected with the R18 labeled virus at ‘0′ min (b), ‘60′ min (c), or PpFucCS-treated and infected with the R18 labeled virus at ‘0′ min (d), ‘5′ min (e), ‘10′ min (f), ‘20′ min (g), ‘40′ min (h), and ‘60′ min (i). (A) Images showing the different groups either treated or mock treated. The cell nuclei appear blue fluorescent while the red fluorescent virus particles are indicated by the circle and arrows. (B) Bar plot represents the number of fluorescent virus particles per image field for treatment groups at different time points. The results are representative of four independent experiments and were analyzed by performing one-way ANOVA test with multiple comparisons (comparing the means of each test with mock-treated control at ‘60′ min). The standard error of the mean was plotted as error bars. The ***, and ns indicate p-value < 0.001, and ≥0.05, respectively.
	Figure 4. Effect of treatment of PpFucCS and its oligosaccharides on cell viability of HFF cells. The primary HFF were pretreated for one hour using PpFucCS and its oligosaccharides at the highest concentration used for inhibition assays (50 μg/mL) along with heparin (UFH) control. The HFFs were either mock infected (A) or infected with HCMV at a MOI of 3.0 (B) in the presence of test compounds. Cells were harvested at 5 days post-infection and cell viability was assessed using a trypan blue exclusion assay. The results are representative of two independent experiments. The standard error of the mean was plotted as error bars. The *, , and ns indicate p-value between 0.001 and 0.01, between 0.01 and 0.05, and ≥0.05, respectively.

References [+] :

Aquino, Glycosaminoglycans and infection. 2016, Pubmed