The VP5 domain of VP4 can mediate attachment of rotaviruses to cells

Some animal rotaviruses require the presence of sialic acid (Sri) on the ce ll surface to infect the cell. We have isolated variants of rhesus rotaviru s (RRV) whose infectivity no longer depends on SA, Both the SA-dependent an d -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8, In this work we have compared the binding characteristics of w ild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactiv e binding assay, both viruses bound to the cells in a saturable and specifi c manner, When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant na r3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not th at of RRV, These results suggest that while RRV binds to the cell through V P8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produ ced in bacteria as fusion products with glutathione S-transferase, were fou nd to bind to MA104 cells in a specific and saturable manner and, when prei ncubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinan t proteins inhibited the infectivity of nar3 and RRV, respectively, confirm ing the results obtained in the binding assays. Interestingly, when the inf ectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the int eraction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.