Integrins α2β1 and α4β1 Can Mediate SA11 Rotavirus Attachment and Entry into Cells

Abstract

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins ␣2␤1 and ␣4␤1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing ␣2␤1 or ␣4␤1 integrins via transfection is increased over virus binding to and infection of cells transfected with ␣3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of ␣2␤1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing ␣2␤1 was elevated over binding to control cells and was specifically blocked by the anti-␣2 monoclonal antibody AK7. Virus growth in ␣4-transfected K562 cells which had also been induced to express ␣2␤1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, ␣2␤1 and ␣4␤1, are capable of acting as cellular receptors for SA11 rotavirus.Rotaviruses, members of the family Reoviridae, are the major etiological agents of severe acute gastroenteritis in infants and young children worldwide and are important pathogens in most mammalian species. It is anticipated that the longawaited introduction of the first vaccine against human rotavirus into use in North America in 1998 will lead to a reduction in the most severe human illness associated with this virus, but other vaccination and therapeutic approaches are required. Novel strategies may be devised following the identification of cellular receptors for rotavirus and from an understanding of the process of viral entry into cells, particularly into intestinal epithelial cells. These are essential steps for productive rotavirus infection and major determinants of host cell tropism.The nonenveloped, icosahedral rotavirus particle consists of a genome of 11 segments of double-stranded RNA (48) in a triple-layered protein capsid (66). The outermost layer of each virion is composed of trimers of the 37-kDa glycoprotein VP7 and 60 spikes of the 88-kDa protein VP4, probably as dimers, which extend about 12 nm above the VP7 surface and interact extensively with VP7 throughout the outer surface (47,65,66).Both VP4 and VP7 independently elicit neutralizing, protective antibodies and are virulence determinants (26, 46). VP4 is an important determinant of host cell tropism (27), receptor binding, and cell penetration (37). Proteolytic cleavage of VP4 into two...

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition

“…To this end, we showed that 8-aa RGD peptides (namely pRGD-Hu, pRGD-Bo, and pRGD-Eq derived from human, bovine, or equine lactadherin, respectively) are endowed with a slight antiviral efficacy. Published studies provide evidence that rotavirus infectivity is not inhibited by RGD peptide, and function-blocking antibodies to RGD-binding integrins do not inhibit rotavirus infection (20,23,53). No evidence for Wa binding to integrins via RGD is available.…”

“…In addition, DGE-containing peptides, such as Asp-Gly-Glu-Ala (DGEA), specifically inhibit rotavirus-cell binding and infection mediated by ␣2␤1 (9,20,21,25). Binding by infectious monkey (SA11 and RRV) and human (Wa) rotaviruses to recombinant ␣2␤1 expressed on K562 cells was specifically inhibited by DG-containing peptides and a function-blocking antibody to the ␣2I domain (9,21,23). Therefore, the interaction of rotavirus with ␣2␤1 integrin can be considered a target for the development of antiviral agents aimed at preventing or reducing rotavirus infection.…”

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

“…Infection by the majority of rotaviruses is independent of terminal SA, but it might involve subterminal SA (14,22). SA11 binding to ␣2␤1 expressed on K562 cells as a result of transfection specifically resulted in increased infectivity, which was inhibited by cellular treatment with an anti-␣2 MAb (23). Cellular receptors for viruses bind virus via virusspecific proteins, and this binding leads to productive cellular infection.…”

“…Integrins ␣2␤1, ␣X␤2, and ␣4␤1 were implicated in group A rotavirus cell attachment and entry (11,23), and ␣V␤3 was proposed to mediate rotavirus cell entry (20). Integrin usage by rotaviruses was discovered when VP5* was shown to contain the type I collagen-derived, ␣2␤1 ligand sequence DGE at amino acids 308 to 310, and the fibrinogen-derived ␣X␤2 integrin ligand sequence GPR was identified in VP7 at amino acids 253 to 255 (11).…”

The effect of concanavalin A on the replication of rotavirus (SA-11) in cell culture

“…Binding of RV in a mixture of nonsialylated glycolipids from bovine brain and mouse intestine was demonstrated suggesting thatattachment and penetration of the virus dependupon different cellular components, and that the former takes place in two stages, dependent and independent of sialylated compounds at cell surface (Willoughby et al, 1990;Srnka et al, 1992;Méndez et al, 1993;Ludert et al, 1996;Isa et al, 1997;Rolsma et al, 1998). The demonstration that VP4 and VP7, respectively present sites for recognition of α2β1 and α4β1, thought to be RV receptors, suggested alternative way for virus penetration; however sugar residues were not considered (Coulson et al, 1997;Hewish et al, 2000). Wheat agglutinin and ConA were previously shown to inhibit several viruses for binding to true receptors or to inhibit progeny maturation and liberation (Ziegler & Pozos, 1981;Okada & Kim, 1972;Poste et al, 1974;Lonberg-Holm 1975;Cartwright, 1977;Khélifa & Menezes, 1982;Conti & Tsiang, 1985).…”