Monkey Rotavirus Binding to α2β1 Integrin Requires the α2 I Domain and Is Facilitated by the Homologous β1 Subunit

Abstract

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-␣2␤1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from ␣2␤1 integrin, and SA11 precipitated ␤1 from ␣2␤1-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 ␣2␤1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human ␣2␤1 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 ␣2␤1 were not essential for rotavirus binding. Rotavirus-␣2␤1 binding led to increased virus infection and RRV growth. SA11 and RRV require the ␣2 I domain for binding to ␣2␤1, 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 ␣2␤1 integrin recognition sequence ...

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

“…This is consistent with the D308 conservation we found here in integrin-using rotaviruses, the abrogation of nar mutant cellular binding by VP5* containing D308A reported previously (55), and the requirement for Asp in the DGEA peptide for inhibition of collagen binding to ␣2␤1 by DGEA (48). It also confirms and extends studies reported elsewhere, which demonstrated by deletion of the ␣2 I domain that this region is necessary for RRV and SA11 binding to human ␣2␤1 expressed on CHO cells (35). As we have shown here that the ␣2 I domain is both necessary and sufficient for VP5* binding to occur, structural studies of rotavirus-␣2␤1 interactions can now be focused on VP5* binding to the ␣2 I domain.…”

Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

“…Expression of the ␣ 2 ␤ 1 -integrin conferred vulnerability to rotavirus infection, suggesting that this integrin served as a rotavirus receptor. Subsequent studies identified the integrins ␣ x ␤ 2 , ␣ v ␤ 3 , and ␣ 4 ␤ 1 as other surface proteins that play a role in rotavirus attachment (13,14,16,21). A survey of the cholangiocyte and hepatocyte cell surface using FACS analysis revealed that cholangiocytes uniquely expressed ␣ 2 ␤ 1 , providing a potential mechanistic basis for RRV tropism to cholangiocytes.…”

“…Cell-surface expression of the integrins ␣ 2 ␤ 1 , ␣ 4 ␤ 1 , ␣ x ␤ 2 , and ␣ v ␤ 3 has been shown to play a role in the attachment and entry of rotaviruses into other cell lines (8,13,14,16,21). Flow cytometry was performed on the mCl and H2.35 cells to determine whether they express the integrin subunits ␣ 1 , ␣ 2 , ␣ 4 , ␣ v , ␣ x , ␤ 1 , ␤ 2 , or ␤ 3 .…”

“…Consistent with the in vivo findings, RRV was better able to replicate in cholangiocytes than hepatocytes. Because the ability of rotavirus to infect cells appears to be regulated by cell-surface expression of the integrins ␣ 2 ␤ 1 , ␣ 4 ␤ 1 , ␣ v ␤ 3 , and ␣ x ␤ 2 , which serve as viral receptors (8,13,14,16,21), the cholangiocyte was surveyed for integrin expression and found to uniquely express the ␣ 2 ␤ 1 -integrin when compared with hepatocytes. Based on this information, we hypothesized that expression of the ␣ 2 ␤ 1 -integrin serves as a determinant governing cholangiocyte susceptibility to RRV infection contributing to the mechanism by which rotavirus initiates the experimental model of biliary atresia.…”

RNAi screen identifies KIF15 as a novel regulator of integrin endocytic trafficking

“…Importantly, the expression of a2b1 integrin is strongly reduced in breast cancer, and a2b1 downregulation correlates with increased cell invasion and metastasis and thus with poor prognosis (Zutter et al, 1998;Ramirez et al, 2011). a2b1 integrin also acts as a co-receptor for several pathogens, such as echovirus 1 (EV1), rotavirus and human cytomegalovirus (Bergelson et al, 1992;Londrigan et al, 2003;Feire et al, 2004;Fleming et al, 2011). Binding of EV1 induces clustering of inactive a2b1 and directs virus-bound a2 integrin to a calpain-dependent degradation pathway in multivesicular bodies (Rintanen et al, 2012;Karjalainen et al, 2008;Upla et al, 2004).…”