Multiplicity of rotavirus receptors

Despite the advances in the molecular and structural biology of these viruses, little is known about the rotavirus cell receptors. A number of glycoconjugates have been shown to bind to, and to block the infectivity of, SA-dependent animal rotavirus strains, and some of them have been suggested to play a role as possible receptors, like GM3 gangliosides in newborn piglet intestine (Rolsma et al 1998), GM1 in LLC-MK2 cells (Superti & Donelli 1991), and 300-330 kDa glycoproteins in murine enterocytes (Bass et al 1991). It has also been suggested that the NA-resistant ganglioside GM1 may act as a receptor for some HRV strains in MA104 cells (Guo et al 1999). Recently, it was reported that VP4 contains the DGE and IDA tripeptide sequence motifs known to interact with integrins a2j1 and a4j1, respectively (Fig. 1), while VP7 contains the aXj2 integrin ligand site GPR, and the a4j1 binding motif LDV (Coulson et al 1997, Hewish et al 2000). Antibodies to the integrin subunits a2, J2 and a4, as well as peptides that mimic the ligand sites were shown to block the infectivity of the SA-dependent rotavirus SA11 and the HRV strain RV5 (Coulson et al 1997). It was also shown that integrins a2j1 and a4j31 can mediate the attachment and entry of rotavirus SA11 into the human myelogenous leukemic cell line K562 (Hewish et al 2000).

As part of the biochemical characterization of the rotavirus cell receptors, we have recently shown that the infectivity of rotaviruses RRV, nar3 and Wa is

TABLE 2 Effect of metabolic inhibitors, cell membrane cholesterol depletion, and octyl-j-glucoside on the infectivity and binding of rotaviruses in MA104 cells

%oInfectivity % Binding

TABLE 2 Effect of metabolic inhibitors, cell membrane cholesterol depletion, and octyl-j-glucoside on the infectivity and binding of rotaviruses in MA104 cells

%oInfectivity % Binding

Inhibitor*

RRV

nar3

Wa

RRV

nar 3

Wa

No treatment

100

100

100

100

100

100

PDMP (25 ^g/ml)

20

40

23

110

46

104

Tunicamycin (2 ^g/ml)

56

48

-

111

101

94

BenzylGalNAc (2 mM)

101

150

147

ND

ND

ND

Octyl-S-glucoside (0.2%)

41

41

39

32

40

33

S-cyclodextrin (10 mM)

9

6

5

112

109

116

OG extractb (20 ^g/ml)

5

3

4

60

59

57

aMA104 cell monolayers were incubated with the indicated concentration of inhibitor for 1 h (j-cyclodextrin), 24 h (tunicamycin), or 72 h (PDMP and BenzylGalNAc) at 37 °C, or for 90min (octyl-j-glucoside) at room temperature, before virus infection.

^Rotaviruses were incubated with either 20 or 400 ^g/ml of OG-extracted proteins, for the binding and infectivity inhibition assays, respectively. At 20 ^g/ml the binding of all three viruses was inhibited by about 40%.

The mean of at least three independent experiments carried out in duplicate is shown.

aMA104 cell monolayers were incubated with the indicated concentration of inhibitor for 1 h (j-cyclodextrin), 24 h (tunicamycin), or 72 h (PDMP and BenzylGalNAc) at 37 °C, or for 90min (octyl-j-glucoside) at room temperature, before virus infection.

^Rotaviruses were incubated with either 20 or 400 ^g/ml of OG-extracted proteins, for the binding and infectivity inhibition assays, respectively. At 20 ^g/ml the binding of all three viruses was inhibited by about 40%.

The mean of at least three independent experiments carried out in duplicate is shown.

Wa polio reo nar3

FIG. 2. Inhibition of rotavirus infectivity by the OG extract. 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, after an adsorption period at 4 °C, the inoculum was removed and the infection was left to proceed for 14 h at 37 °C. At this time the cells were fixed, and the infectious titre was determined by an immunoperoxidase focus assay. Percentage infectivity is referred to the infectivity of the viruses incubated in 0.2% OG. Error bars represent one standard error of the mean of three independent experiments carried out in duplicate.

Wa polio reo nar3

100 200 300 400 OG-extracted protein (/jg/ml)

FIG. 2. Inhibition of rotavirus infectivity by the OG extract. 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, after an adsorption period at 4 °C, the inoculum was removed and the infection was left to proceed for 14 h at 37 °C. At this time the cells were fixed, and the infectious titre was determined by an immunoperoxidase focus assay. Percentage infectivity is referred to the infectivity of the viruses incubated in 0.2% OG. Error bars represent one standard error of the mean of three independent experiments carried out in duplicate.

partially blocked by metabolic inhibitors of N-glycosylation (tunicamycin), and glycolipid synthesis (PDMP), while it is not affected by the inhibition of the cellular O-glycosylation (Guerrero et al 2000a). In addition, we also showed that depletion of cholesterol from the cell membrane with methyl-^-cyclodextrin reduced the infectivity of the three viruses by more than 90%, while not affecting their binding to the cell (Table 2). The involvement of N-glycosylated proteins, glycolipids, and cholesterol in rotavirus infection suggest that the virus receptor(s) might be forming part of the cell membrane glycosphingolipid-enriched lipid microdomains, termed rafts (Simons & Ikonen 1997).

In a different approach we showed that treatment of MA104 cells with the non-ionic detergent octyl-^-glucoside (OG), under non-lytic conditions, renders the cells largely refractory to binding and infection by rotaviruses (Table 2) (Guerrero et al 2000a), most probably due to the extraction of the rotavirus receptor(s). Accordingly, pre-incubation of the viruses with the OG extract inhibited infectivity by more than 95% (Fig. 2). Five protein bands with the ability to block rotavirus infectivity were purified by preparative electrophoresis from these extracts, and amino acid sequence analysis of the band of 110 kDa, revealed the presence, among other proteins, of the ^3 integrin subunit.

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