Cell surface ligands for rotavirus: Mouse intestinal glycolipids and synthetic carbohydrate analogs

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143

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAc␣3-Gal␤) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGc␣3Gal␤ is preferred to NeuAc␣3Gal␤. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

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confidence: 99%

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confidence: 99%

Glycosphingolipid Binding Specificities of Rotavirus: Identification of a Sialic Acid-Binding Epitope

Delorme

Brüssow

Sidoti

et al. 2001

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143

“…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).…”

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confidence: 99%

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

Londrigan

Graham

Takada

et al. 2003

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 ...

“…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).…”

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confidence: 99%

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

Ciarlet

Crawford

Cheng

et al. 2002

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 (␣2␤1) 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 (CHO␣2) or with both the human ␣2 and ␤1 subunits (CHO␣2␤1) 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, CHO␣2, and CHO␣2␤1 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.