The SA binding domain of the virus is located on the tip of VP8 (Dormitzer et al., 2002; Isa et al., 1997). The crystal structure of several VP8 proteins has been resolved (Dormitzer et al., 2002; Blanchard et al., 2007; Monnier et al., 2006; Yu et al., 2011); this protein has a galectin-like fold with two β-sheets separated by a shallow cleft. The crystal structures of VP8 of NA-sensitive rotavirus strains complexed with SA showed that SA binds near the cleft region. Interestingly, when the VP8 proteins of NA-resistant strains were analyzed, this cleft was found to be wider (Haselhorst et al., 2009). Even though the structure of a human VP8 with a wider cleft in complex with a glycan has not been reported, NMR and modeling studies propose that a wider cleft would allow binding of gangliosides with internal SA (Venkataram Prasad et al., 2014).

Gangliosides and glycosphingolipids having one or more SA residues, are a large and heterogeneous family of lipids present on the extracellular leaflet of mammalian plasma membranes, and have been associated with rotavirus cell entry for some time. Using a thin-layer chromatography binding assay, NA-sensitive rotavirus strains were found to bind to gangliosides with terminal SA, while the NA-resistant strains tested interacted with gangliosides containing subterminal SA (Delorme et al., 2001). The role of gangliosides during the infection of MA104 cells by NA-sensitive and -resistant strains was also analyzed after silencing by RNA interference the expression of two key enzymes involved in ganglioside synthesis: UDP-glucose:ceramide glucosyltransferase and lactosyl ceramide-α-2,3-sialyl transferase 5 (Martinez et al., 2013). Knocking down the expression of these enzymes decreased ganglioside levels, resulting in a diminished infectivity of all rotavirus strains tested. Interestingly, the binding of virus to cells with low ganglioside levels was not affected, suggesting that the rotavirus-ganglioside interaction is not necessary for cell surface binding, but during a later step of the cell entry process.

Recently, it was demonstrated that many human rotavirus strains bind to human histo-blood group antigens (HBGAs). Some of them bind H-type glycans, while others have A-type glycan specificity (Bohm et al., 2015; Hu et al., 2012; Huang et al., 2012; Liu et al., 2012) (Table 2.2.1). In addition, the VP8 of a P[11] neonatal strain, was shown to specifically bind a precursor of the H type II HBGA, which forms the core structure of type II glycans (Ramani et al., 2013). The crystal structure of a [P14] VP8 showed that it has a narrow cleft, like that present in NA-sensitive strains, and that the A-type HBGA binds in the same location in the cleft as SA in the animal VP8 structure (Venkataram Prasad et al., 2014). Of interest, glycan modification is thought to vary during neonatal development, which could explain the age-restricted infectivity of neonatal rotavirus strains. It is also of interest that the HBGA phenotype and the secretory status of children seem to correlate with the VP4 genotype of the infecting rotavirus strain (Nordgren et al., 2014; Van Trang et al., 2014).

The interaction of rotavirus with integrin α2β1 is mediated by a DGE motif located towards the amino-terminal end of the VP5 domain of VP4, while the domain I of the integrin subunit α2 is involved in this interaction (Graham et al., 2003; Zarate et al., 2004). The amino acid residues on the I domain that interact with the virus spike protein were identified by expressing α2β1 integrin mutants in CHO cells (Fleming et al., 2011). On the other hand, integrin αVβ3 interacts with rotavirus through a linear sequence in VP7 (Graham et al., 2003; Zarate et al., 2004).

A cellular molecule used by all rotavirus strains is hsc70 (Gutierrez et al., 2010; Guerrero et al., 2002). The interaction between the viral particle and hsc70 is mediated by VP5, through a domain located between amino acids 642 and 659 (Zarate et al., 2003). Since a synthetic peptide corresponding to this region blocks virus infectivity but not cell binding, it has been suggested that the interaction between the virus and hsc70 is at a postattachment step. Apparently the ATPase domain of hsc70 is involved in promoting conformational changes in the viral particle that facilitate virus entry (Perez-Vargas et al., 2006).

Additionally, some of the molecules that participate in attachment and postattachment interactions (gangliosides, integrins α2β1, αVβ3, and hsc70) group together in detergent-resistant membrane domains (lipid rafts), where infectious viral particles are also present during cell infection (Isa et al., 2004). The integrity of these domains is fundamental for viral infection, since their destabilization severely decreases the infectivity of all rotavirus strains tested (Gutierrez et al., 2010; Guerrero et al., 2000) (Fig. 2.2.1A).

Rotaviruses are classical gastrointestinal viruses that infect the mature enterocytes located at the tip of the small intestinal villi. Given the basolateral localization of integrins in these cells, an important question is how rotaviruses reach these cell receptors to enter the cell. One explanation was offered when it was shown that a recombinant VP8 protein of RRV is able to decrease the transepithelial electrical resistance of polarized Madin–Darby canine kidney (MDCK) cells (Nava et al., 2004). Furthermore, this VP8 protein was shown to open the tight junctions, releasing basolateral proteins (integrins αVβ3, β1, and the Na⁺-K⁺-ATPase) to the apical side of the cells (Nava et al., 2004). In addition, it has recently been found that the tight-junction proteins JAM-A, occludin, and ZO-1 are important for the entry of some rotavirus strains (Torres-Flores et al., 2015).

It remains to be established whether all the described molecules work in concert, or represent alternative routes of entry. However, it is noteworthy that the assays used to block the interaction of rotaviruses with each of these proposed receptors and coreceptors using different approaches, such as proteases, antibodies, peptides, sugar analogues, or siRNAs, only decrease viral infectivity by less than 10-fold, suggesting that either a more relevant entry factor for rotavirus has yet to be found, the virus can use more than one route of entry, or the cellular factors that allow the entry of rotavirus are redundant.