Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infectionby simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the ␣2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-␣21 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from ␣21 integrin, and SA11 precipitated 1 from ␣21-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160-to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the ␣2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human ␣2 or ␣21. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human ␣21 and, to a lesser extent, human ␣2 combined with hamster 1. Binding was inhibited by anti-␣2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to ␣2, and required the presence of the ␣2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the ␣2 I domain that are necessary for type I collagen binding to ␣21 were not essential for rotavirus binding. Rotavirus-␣21 binding led to increased virus infection and RRV growth. SA11 and RRV require the ␣2 I domain for binding to ␣21, and their binding to this integrin is distinguishable from that of collagen.Virus attachment and entry into host cells are multistep processes that influence cellular tropism and can involve sequential recognition of multiple receptors and coreceptors. Rotaviruses, a genus within the Reoviridae family, cause severe gastroenteritis following infection of intestinal enterocytes. The virus spike protein, VP4, which is a major determinant of tropism and receptor binding (4,20,51,58), is proteolytically cleaved by trypsin into VP5* and VP8*, which increases the virus infectivity and internalization rate (1,14,28,29). Several glycoconjugates have been implicated in rotavirus attachment (4,5,22,32,38,42,68,74,84). Although a minority of animal rotaviruses, including simian strains SA11 and RRV, can utilize terminal sialic acids (SA) as receptors (12, 13, 22, 32), SA are not essential for infectivity (63). SA-using porcine rotaviruses OSU and CRW-8 appear to use ganglioside-and glycolipid-based receptors, respectively (43, 68). RRV binds sialosides with low affinity via a galectin-like region in VP8* (24,25).In searching for rotavirus receptors, Coulson et al. found that VP4 and rotavirus outer capsid protein VP7 contain sequences corresponding to integrin recognition sites (17). Integrins are ␣/ heterodimeric, transmembrane glycoproteins important for cell surface adhesion and signaling. The RDGE sequence in VP4 at amino acids (aa) 307 to 310 corresponds to the putative ␣21 integrin recognition sequence ...