Abstract
The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.
Highlights
Integrins are heterodimers of ␣ and  subunits anchored in the cell membrane that interact with the extracellular matrix, the actin cytoskeleton and signaling pathways to regulate cell processes including survival, differentiation, and migration [1]
The DGE sequence is externally located on the trypsin-primed and putative post-penetration structural forms of VP5* [32, 33]. These findings demonstrate the importance of ␣2I in monkey rotavirus recognition of ␣21
Cell Lines—The derivation, mutagenesis, and propagation with G418 sulfate selection (Invitrogen) of CHO cells transfected with cDNA encoding empty vector (PBJ-1); human ␣2 combined with hamster 1 (Hu ␣2); human ␣2 containing a single point mutation or deletion, combined with hamster 1 (n ϭ 51); human ␣2 with a human-to-mouse swap at amino acids 212–216 in ␣2I, combined with hamster 1 (Hu-mur ␣2I); human ␣2 containing a human-to-mouse swap of ␣2I, combined with hamster 1 (Mur ␣2I); human ␣2 combined with human 1 (Hu ␣21); and human ␣2 and human 1 with a D130A mutation (Hu ␣21 D130A) were as described previously (17, 34 –36)
Summary
Cell Lines—The derivation, mutagenesis, and propagation with G418 sulfate selection (Invitrogen) of CHO cells transfected with cDNA encoding empty vector (PBJ-1); human ␣2 combined with hamster 1 (Hu ␣2); human ␣2 containing a single point mutation or deletion, combined with hamster 1 (n ϭ 51); human ␣2 with a human-to-mouse swap at amino acids 212–216 in ␣2I, combined with hamster 1 (Hu-mur ␣2I); human ␣2 containing a human-to-mouse swap of ␣2I, combined with hamster 1 (Mur ␣2I); human ␣2 combined with human 1 (Hu ␣21); and human ␣2 and human 1 with a D130A mutation (Hu ␣21 D130A) were as described previously (17, 34 –36). Antibodies and Rotavirus Strains—Anti-human ␣2 monoclonal antibodies AK7 [37, 38] and HAS4 [39], and isotype control antibodies MOPC21 and UPC10, were obtained as described previously [7, 40]. Rotavirus infectivity titers were determined by indirect immunofluorescent staining of MA104 cells inoculated with serial dilutions of samples followed by microscopy, as described previously [8, 17]. Flow Cytometric Analysis—Surface ␣21 integrin expression was detected on CHO cell lines by direct (Ha1/29) or indirect (AK7, Gi9, Has4) immunofluorescent staining of 8 ϫ 105 cells followed by flow cytometry as described previously [8]. Rotavirus titers (expressed in FCFU/ml) were determined by titration in MA104 cells as described above. Data were analyzed by Student’s t test unless otherwise indicated
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