Binding of Sindbis virus to cell surface heparan sulfate - PubMed (original) (raw)

Binding of Sindbis virus to cell surface heparan sulfate

A P Byrnes et al. J Virol. 1998 Sep.

Abstract

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.

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Figures

FIG. 1

Inhibitory effects of polysaccharides on plaque formation by SV. SV strain Toto 1101 was used throughout this study. (A) SV was mixed with various GAGs or the highly sulfated polysaccharide dextran sulfate and incubated on monolayers of BHK cells for 1 h at 37°C before being overlaid with agar. (B) The requirement for high sulfation was shown by the inability of HS or various forms of desulfated heparin to inhibit SV plaque formation. Abbreviations: CDSNS, completely desulfated, N sulfated; CDSNAc, completely desulfated, N acetylated; NDSNAc, N desulfated, N acetylated. Error bars are omitted for clarity of presentation. Each point is the mean of three or more measurements. ∗, P of <0.05 versus no inhibitor.

FIG. 2

Digestion of GAGs decreases the ability of cells to bind SV. CHO monolayers were predigested with various concentrations of heparinase I or chondroitinase ABC, and the amount of radiolabeled virus bound after 1 h at 4°C was measured. Each point is the mean of three measurements. ∗, P of <0.05 versus no enzyme.

FIG. 3

Binding of alphaviruses to GAG-deficient cells. (A) Binding of SV to wild-type CHO cells, pgsE cells with partially desulfated HS, pgsD cells lacking HS but having elevated CS, and pgsA cells with no HS or CS. At each time point, binding to CHO cells was significantly greater than binding to the three mutant cell lines. Binding to pgsE cells was also significantly greater than binding to pgsA and pgsD cells. There was no significant difference between binding to pgsD and binding to pgsA cells. (B) Binding of RRV. Binding to CHO cells was significantly greater than to the three mutant cell lines (but see text). Each point is the mean of three measurements.

FIG. 4

Binding of alphaviruses to immobilized GAGs. Ninety-six-well plates were coated with various GAGs, and the amount of radiolabeled virus that bound was assessed. (A) Binding of SV. (B) Binding of RRV. Ten thousand counts per minute of virus per well were used. Each bar represents the mean of four measurements. ∗, P of <0.05 versus uncoated wells. See the legend to Fig. 1 for abbreviations.

FIG. 5

Antibodies against the E2 glycoprotein can inhibit binding of SV to heparin. Ninety-six-well plates coated with heparin were incubated with 5,000 cpm of SV in the presence or absence of IgG or Fab fragments. R6 recognizes the E2c epitope, and 202 recognizes the E2ab epitope. 3E1 is a control antibody against HSV-1. Each point is the mean of two measurements. An additional experiment gave similar results.

FIG. 6

Elution of alphavirus from heparin-Sepharose columns. Virus (70,000 to 100,000 cpm) was added to the column in 100 mM NaCl buffer and eluted with a gradient of from 100 to 500 mM NaCl. (A) SV. (B) RRV. Single viral clones from RRV peak fractions 10 and 17 were selected, grown, and radiolabeled. These viral clones eluted at salt concentrations similar to the peaks from which they were originally taken (C and D).

FIG. 7

Effect of overlay on plaque diameter. Virus was absorbed to monolayers of BHK cells for 1 h at 37°C and then overlaid with 0.6% agar or agarose with or without additives. Plaque diameters were measured at 2 days to the nearest 0.5 mm. (A) Effect of overlay composition on SV strain Toto 1101. ∗, P of <0.05 versus agar plus DEAE-dextran; †, P of <0.05 versus agarose. (B) Overlay composition had no significant effect on the plaque size of RRV strain T48. Each bar is the mean of 15 or more measurements. Results were analyzed by ANOVA on ranks, followed by Dunn’s test.

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Binding of Sindbis virus to cell surface heparan sulfate - PubMed (original) (raw)