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

In an attempt to identify the rotavirus receptor, we tested 46 cell lines of different species and tissue origins for susceptibility to infection by three N-acetyl-neuraminic (sialic) acid (SA)-dependent and five SA-independent rotavirus strains. Susceptibility to SA-dependent or SA-independent rotavirus infection varied depending on the cell line tested and the multiplicity of infection (MOI) used. Cells of renal or intestinal origin and transformed cell lines derived from breast, stomach, bone, or lung were all susceptible to rotavirus infection, indicating a wider host tissue range than previously appreciated. Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), guinea pig colon (GPC-16), rat small intestine (Rie1), and mouse duodenum (MODE-K) cells were found to support only limited rotavirus replication even at MOIs of 100 or 500, but delivery of rotavirus particles into the cytoplasm by lipofection resulted in efficient rotavirus replication. The rotavirus cell attachment protein, the outer capsid spike protein VP4, contains the sequence GDE(A) recognized by the VLA-2 (21) integrin, and to test if VLA-2 is involved in rotavirus attachment and entry, we measured infection in CHO cells that lack VLA-2 and CHO cells transfected with the human 2 subunit (CHO2) or with both the human 2 and 1 subunits (CHO21) of VLA-2. Infection by SA-dependent or SA-independent rotavirus strains was 2- to 10-fold more productive in VLA-2-expressing CHO cells than in parental CHO cells, and the increased susceptibility to infection was blocked with anti-VLA-2 antibody. However, the levels of binding of rotavirus to CHO, CHO2, and CHO21 cells were equivalent and were not increased over binding to susceptible monkey kidney (MA104) cells or human colonic adenocarcinoma (Caco-2, HT-29, and T-84) cells, and binding was not blocked by antibody to the human 2 subunit. Although the VLA-2 integrin promotes rotavirus infection in CHO cells, it is clear that the VLA-2 integrin alone is not responsible for rotavirus cell attachment and entry. Therefore, VLA-2 is not involved in the initial attachment of rotavirus to cells but may play a role at a postattachment level. Group A rotaviruses are important viral agents of acute gastroenteritis in young children and many animal species (24). 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. This restricted tissue- and cell-type-specific tropism is mediated by one or more specific host cell surface receptors that mediate rotavirus attachment. Numerous studies have implicated glycoconjugates (glycoproteins, glycolipids, and glycosphingolipids) as the putative rotavirus receptor(s) (4, 19, 32, 34, 41, 55, 58, 59, 66). A minority of animal rotaviruses require the presence of N-acetyl-neuraminic (sialic) acid (SA) residues on the cell surface for efficient binding and infectivity, but most animal and human rotaviruses are SA independent (13). Binding of rotaviruses to SA residues has been mapped to amino acids 93 to 208 of the VP4 cleavage product VP8* (38). However, the isolation of SA-independent animal rotavirus strain variants from SA-dependent strains that are efficient in attachment and infection confirms that