Biochemical characterization of rotavirus receptors in MA104 cells

Abstract

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 beta-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-beta-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.

FIG. 1

(A) Recovery of the susceptibility of MA104 cells to rotavirus infection after extraction with OG. Cell monolayers in 96-well plates were extracted with 0.2% OG and allowed to recover in MEM at 37°C. At the indicated times, the monolayers were washed with PBS and infected with rotaviruses. (B) Inhibition of rotavirus infectivity by the OG extract from MA104 cells. The indicated concentrations of OG-extracted protein were incubated with the viruses for 90 min at 37°C. The virus-protein mixtures were used to infect MA104 cell monolayers in 96-well plates. In both panels, the percent infectivity is relative to the infectivity of the viruses incubated in 0.2% OG. Error bars represent 1 standard error of the mean of three or more experiments carried out in duplicate.

FIG. 2

Inhibition of rotavirus infectivity by OG extracts from cells poorly permissive to rotavirus infection. OG-extracted proteins (20 μg/ml) from CHO, BHK, L, or MA104 cells (as indicated) were incubated with the viruses for 90 min at 37°C. The virus-protein mixtures were used to infect MA104 cell monolayers in 96-well plates. The percent infectivity is relative to the infectivity of the viruses incubated in 0.2% OG. Error bars represent 1 standard error of the mean of three experiments carried out in duplicate.

FIG. 3

Biochemical nature of the inhibitory factor present in the OG extract. A 0.2% OG extract was obtained from cells in suspension. Just prior to the incubation with the virus, the extract was either boiled (95°C) for 15 min (heat), incubated with 2 mg of trypsin per ml of extract for 1 h at 37°C (trypsin), or incubated with 36 mU of neuraminidase per ml (NA). The untreated extract (no treatment) was used as a positive control. Viruses and extract (100 μg of protein extract per ml of virus) were mixed and incubated for 90 min at 37°C, and then MA104 cells in 96-well plates were infected with the virus-protein mixtures. The percent infectivity is relative to the infectivity of viruses incubated with a solution of 0.2% OG in MEM (virus control). Error bars represent 1 standard error of the mean of three or more experiments carried out in duplicate.

FIG. 4

Analysis of the proteins extracted from MA104 cells. Cell monolayers were treated with either neuraminidase, tunicamycin, or PDMP, as described in Materials and Methods, and the cells were then extracted with 0.2% OG for 90 min at room temperature. (A) The extracted proteins were separated by electrophoresis under reducing conditions in an SDS–11% polyacrylamide gel and silver stained. OG-extracted proteins from MA104 cells treated with neuraminidase (lane 2), tunicamycin (lane 3), or PDMP (lane 4) or left untreated (lane 5) are shown. Lane 1 contains molecular mass markers. (B) Cells in suspension were extracted with 10 mM β-cyclodextrin for 1 h at 37°C, as described in Materials and Methods. Proteins in untreated cells (lane 1), extracted cells (lane 2), and the cyclodextrin extract (lane 3) were analyzed by gel electrophoresis.

FIG. 5

Inhibition of rotavirus infectivity by OG-extracted proteins fractionated by gel electrophoresis. About 250 μg of proteins extracted with 0.2% OG from MA104 cells was separated by preparative SDS-polyacrylamide gel electrophoresis under nonreducing conditions. After electrophoresis, the gel was stained with Coomassie blue in water, gel slices were cut out, and the proteins were eluted. (A) Inhibitory activity of the eluted proteins present in the fractions shown in panel B. (B) Gel electrophoresis of the eluted protein fractions. Only the portion of the gel where inhibitory activity was found is shown; the remaining higher- and lower-molecular-mass protein fractions had no inhibitory activity.

FIG. 6

Inhibitory activity of hyperimmune sera to OG-extracted proteins. (A and B) OG protein fractions 6 and 10 shown in Fig. 5B, containing polypeptides of around 57 and 75 kDa, respectively, were used to raise antibodies in rabbits. Serial dilutions of the preimmune (dashed lines) and hyperimmune (continuous lines) sera to the 75-kDa protein fraction were incubated with untreated (A) or neuraminidase (NA)-treated (B) MA104 cells for 90 min at 37°C before addition of the virus. Similar inhibition results were obtained with the serum to the 57-kDa protein fraction (data not shown). Error bars represent 1 standard error of the mean of three or more experiments carried out in duplicate. (C) Immunoblot analysis of the OG-extracted proteins. The proteins extracted from MA104 cells with 0.2% OG were separated in an SDS–11% polyacrylamide gel under reducing conditions and transferred to nitrocellulose. The transferred proteins were incubated with a 1,000-fold dilution of the preimmune (lanes 1 and 2) or hyperimmune (lanes 3 and 4) sera to the 57-kDa (lanes 2 and 4) or 75-kDa (lanes 1 and 3) protein fractions. The bound antibodies were developed by incubation with protein A-peroxidase and a chromogenic substrate.

FIG. 7

Immunofluorescence of cells incubated with the serum to the 75-kDa OG protein fraction. MA104 cells were fixed with paraformaldehyde and permeabilized with Triton X-100 (B and D) or not permeabilized (A and C). The cells were incubated with a 1:1,500 dilution of the preimmune (C and D) or hyperimmune (A and B) sera to the 75-kDa protein fraction for 90 min at 37°C and stained with a goat anti-rabbit immunoglobulin G coupled to fluorescein isothiocyanate.

FIG. 8

Isolated proteins with inhibitory activity for rotavirus infectivity. The protein bands that were shown to block rotavirus infectivity after three rounds of purification by preparative gel electrophoresis (see Table 4) were analyzed in an 11% polyacrylamide gel under reducing conditions. The protein bands were detected by silver staining.