Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase from rat liver

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro , identified molecular determinants of glycan binding, and analyzed infection in vivo . We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo , CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo .

Section: Resultsmentioning

confidence: 99%

Murine Noroviruses Bind Glycolipid and Glycoprotein Attachment Receptors in a Strain-Dependent Manner

Taube

Perry

McGreevy

et al. 2012

“…Two inhibitors were used: tunicamycin, which blocks an early step in the N-glycosylation pathway involving transfer between UDPGlcNAc and dolichol-1-phosphate (12), and benzylGalNAc, which is a competitive inhibitor of the transferase (N-acetyl-␣-D-galactosaminyltransferase) involved in the first step of the biosynthesis of most types of O-linked carbohydrates (5). In addition, we used the synthetic analog of ceramide, PDMP, to inhibit the biosynthesis of the glycosphingolipid precursor glucosylceramide (45).…”

Section: Inhibitors Of N Glycosylation and Glycolipid Synthesis Blockmentioning

confidence: 99%

Biochemical Characterization of Rotavirus Receptors in MA104 Cells

Guerrero¹,

Zárate²,

Corkidi

et al. 2000

102

We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with ␤-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-␤-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.

“…2 and 3). As branched chains containing ␣(2,6)-linked sialic acid are not normally found in N-linked glycoproteins, we believe that Nlinked carbohydrate chains containing terminal ␣(2,6)-and ␣(2,3)-linked sialic acid are sufficient to mediate virus uptake (3,5,21).…”

mentioning

confidence: 96%

Direct Correlation between Sialic Acid Binding and Infection of Cells by Two Human Polyomaviruses (JC Virus and BK Virus)

Dugan¹,

Gasparovic²,

Atwood

2008

For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both ␣(2,3)-linked and ␣(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.