Biochemical characterization of rotavirus receptors in MA104 cells

We have tested the effect of metabolic inhibitors, membrane cholesterol dep letion, and detergent extraction of cell surface molecules on the susceptib ility of MA104 cells to infection by rotaviruses. Treatment of cells with t unicamycin, an inhibitor of protein N glycosylation, blocked the infectivit y of the SA-dependent rotavirus RRV and its SA-independent variant nan by a bout 50%, while the inhibition of O glycosylation had no effect. The inhibi tor of glycolipid biosynthesis d,l-threo-1-phenyl-2-decanoylamino-3-morphol ino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human r otavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with beta-cyclodextrin reduced the infectivity of the three virus es by more than 90%. The involvement of N-glycoproteins, glycolipids, and c holesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells in cubated with the nonionic detergent octyl-beta-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to whic h they became refractory to infection, suggesting that OC extracts the pote ntial virus receptor(s) from the cell surface, Accordingly, when preincubat ed 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, in dicating the protein nature of the inhibitor. Two protein fractions of arou nd 57 and 75 kDa were isolated from the extract, and these fractions were s hown to have rotavirus-blocking activity. Also, antibodies to these fractio ns efficiently inhibited the infectivity of the viruses in untreated as wel l as in neuraminidase-treated cells. Five individual protein bands of 30, 4 5, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were final ly isolated from the OG extract. These proteins are good candidates to func tion as rotavirus receptors.