The VP5 Domain of VP4 Can Mediate Attachment of Rotaviruses to Cells

Abstract

Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -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 wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific 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 nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidasetreated 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 interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.

The rotavirus surface protein VP8 modulates the gate and fence function of tight junctions in epithelial cells

“…The RRV VP5 fragment (VP4 gene nucleotides 749 to 2347) was cloned in pGEX-4T-2 (Pharmacia) as previously described (Zarate et al, 2000b). Expression and purification of fusion proteins was performed following previously described standard procedures (Frangioni and Neel, 1993).…”

“…SA-dependent rhesus rotaviruses (RRV) initially bind to the cell through VP8 (Fiore et al, 1991;Isa et al, 1997), whereas variants of RRV that no longer depend on the presence of SA (e.g. nar3), interact with the cell through VP5 (Zarate et al, 2000b).…”

VLA-2 (α2β1) Integrin Promotes Rotavirus Entry into Cells but Is Not Necessary for Rotavirus Attachment

“…Two surface proteins, VP4 and VP7, are present on the outer capsid of rotaviruses, and trypsin cleavage of the spike protein VP4 into VP5* and VP8* enhances rotavirus infectivity and is involved in the early interaction of the virus with the cell (18,67). Rotaviruses infect the mature enterocytes of the villus epithelium of the small intestine.…”

Attachment and Infection to MA104 Cells of Avian Rotaviruses Require the Presence of Sialic Acid on the Cell Surface

“…It has been reported that recombinant VP8 protein, produced in bacteria as a fusion product with glutathione Stransferase (GST-VP8), was found to bind to MA104 cells in a specific and saturable manner and that it was capable of inhibiting the binding of a homologous virus when it was preincubated with MA104 cells [24]. To confirm the specificity of binding of an avian rotavirus to sialic acid on the cell surface, we prepared GST-VP8 of strain PO-13 and carried out a binding assay using purified GST-VP8.…”

“…There is evidence that rotaviruses have multiple plasma membrane receptors, including sialic acid (SA) [6], integrins [4] or other membrane proteins [13]. Some animal rotaviruses can bind to the cell either through interactions mediated by VP8 or VP5 via SA-containing and SA-independent cell surface receptors, respectively [7,24]. Human strains appear to use an SA-independent route [6], and an α2β1 integrin-binding motif (DGE) present in VP5 at amino acids 308-310 may function as the receptor-binding site [23].…”