Integrin 3 functions as a coreceptor for rotaviruses

The relevance of ¡3 integrin for rotavirus infection was established by the fact that antibodies to this integrin subunit reduced by 50% the infectivity of RRV, nar3 and Wa rotaviruses. In accordance to this finding, when vitronectin, a ¡3 integrin ligand, was pre-incubated with cells, it specifically blocked rotavirus infectivity up to 70% (Guerrero et al 2000b).

Since integrins a2^1, a4^1 and aX^2 have been suggested to play a role during rotavirus entry (Coulson et al 1997), we performed blocking experiments using mixes of antibodies directed to these integrins and to aV^3. A clear additive blocking effect was found when antibodies to integrins a2^1 and aV^3 were mixed, suggesting that these two integrins might be involved in different stages of rotavirus infection (Guerrero et al 2000b).

The expression of ¡3 integrin into the poorly permissive CHO cells was shown to facilitate the infectivity of rotaviruses. CHO cells stably transfected with the ¡3 integrin gene (Diaz-Gonzalez et al 1996), overexpressing either

FIG. 3. Effect of antibodies to the cell surface, and of a VP4 peptide, on the binding of RRV and nar3 viruses. MA104 cells were preincubated for 1 h at 37 °C with a MAb to integrin subunit a2, with MAb 2D9 or with peptide DGE. After incubation, these cells were washed, and purified RRV or nar3 viral particles or affinity purified GST-VP8 and GST-VP5 fusion protein were adsorbed for 60min at 4 °C with gentle shaking. The amount of cell bound virus, or fusion protein, was determined by an ELISA, as described (Zarate et al 2000a). The VP4 synthetic peptide evaluated comprises amino acid residues 300 to 321 of the protein, and contains the DGE sequence binding motif for integrin a2j81. Data are expressed as the percentage of virus or recombinant protein binding, in the absence of antibodies or peptide. The arithmetic means and standard deviations of two independent experiments are shown.

FIG. 3. Effect of antibodies to the cell surface, and of a VP4 peptide, on the binding of RRV and nar3 viruses. MA104 cells were preincubated for 1 h at 37 °C with a MAb to integrin subunit a2, with MAb 2D9 or with peptide DGE. After incubation, these cells were washed, and purified RRV or nar3 viral particles or affinity purified GST-VP8 and GST-VP5 fusion protein were adsorbed for 60min at 4 °C with gentle shaking. The amount of cell bound virus, or fusion protein, was determined by an ELISA, as described (Zarate et al 2000a). The VP4 synthetic peptide evaluated comprises amino acid residues 300 to 321 of the protein, and contains the DGE sequence binding motif for integrin a2j81. Data are expressed as the percentage of virus or recombinant protein binding, in the absence of antibodies or peptide. The arithmetic means and standard deviations of two independent experiments are shown.

aIIbß3 or aVß3 integrins, were three to four times more susceptible to rotavirus infection than the parental CHO cell line. This increase in infectivity was shown to be blocked by incubation of the cells with either MAbs to ß3 or vitronectin (Fig. 4) (Guerrero et al 2000b). Furthermore, it was shown that the interaction of rotaviruses with aVß3 is at a postattachment step, probably penetration, since vitronectin and antibodies to ß3 do not, or only slightly, inhibit rotavirus cell attachment. Also, the interaction of rotaviruses with ß3 integrin was found to be RGD-independent, as expected from the fact that neither VP4 nor VP7 have this integrin binding motif (Guerrero et al 2000b).

FIG. 4. The expression of P3 integrin in CHO cells facilitates rotavirus cell infection. Monolayers of control CHO cells or CHO cells expressing integrin aIIb^3 (Diaz-Gonzalez et al 1996), in 96-well plates, were infected with 2xl03 ffu's of RRV, nar3 or Wa viruses per well. After 60 min adsorption at 37 °C, the infection was left to proceed for 16 h at 37 °C, at which time the cells were fixed and immunostained. In the condition where the cells were preincubated with vitronectin (CHO/aIIb^3 + Vn), the integrin ligand (1.5 ^g/ml) was added for 1 hat 37 °C before virus infection. Data are expressed as percentage of the virus infectivity obtained in the CHO cells. The arithmetic mean from two independent experiments performed in duplicate are shown. The standard error is shown.

RRV nar3 Wa

FIG. 4. The expression of P3 integrin in CHO cells facilitates rotavirus cell infection. Monolayers of control CHO cells or CHO cells expressing integrin aIIb^3 (Diaz-Gonzalez et al 1996), in 96-well plates, were infected with 2xl03 ffu's of RRV, nar3 or Wa viruses per well. After 60 min adsorption at 37 °C, the infection was left to proceed for 16 h at 37 °C, at which time the cells were fixed and immunostained. In the condition where the cells were preincubated with vitronectin (CHO/aIIb^3 + Vn), the integrin ligand (1.5 ^g/ml) was added for 1 hat 37 °C before virus infection. Data are expressed as percentage of the virus infectivity obtained in the CHO cells. The arithmetic mean from two independent experiments performed in duplicate are shown. The standard error is shown.

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