Integrins ␣21, ␣X2, and ␣V3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the ␣21 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the ␣X2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of ␣21, ␣X2, and ␣V3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound ␣21, and VP7 interacted with ␣X2 and ␣V3 at a postbinding stage. DGEA inhibited rotavirus binding to ␣21 and infectivity, whereas GPRP binding to ␣X2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed ␣2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the ␣2 I domain. In a novel process, integrin-using viruses bind the ␣2 I domain of ␣21 via DGE in VP4 and interact with ␣X2 (via GPR) and ␣V3 by using VP7 to facilitate cell entry and infection.Rotaviruses are leading causes of acute gastroenteritis in human infants and young animals. Their restricted tropism suggests that very specific virus-host cell interactions are necessary to establish infection. The viral spike protein VP4, the major cell attachment protein, is cleaved by trypsin for enhanced infectivity into two subunits, VP5* (60 kDa) and VP8* (28 kDa). VP4 and the major outer capsid protein VP7 independently define serotype specificities. The inner capsid protein VP6 contains group-specific antigenic determinants. Reassortant rotaviruses containing combinations of the 11 double-stranded RNA genes from two parental viruses can be generated (27) which occasionally show unexpected phenotypes due to VP4-VP7 interactions (43).Integrins ␣21, ␣X2, and ␣41 were implicated in group A rotavirus cell attachment and entry (11, 23), and ␣V3 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, ␣21 ligand sequence DGE at amino acids 308 to 310, and the fibrinogen-derived ␣X2 integrin ligand sequence GPR was identified in VP7 at amino acids 253 to 255 (11). Peptides containing these viral integrin ligand sequences (GPRP and RDGEE), monoclonal antibodies (MAbs) to ␣21 and MAbs to 2, inhibited the infection of monolayers of MA104 and Caco-2 cells by simian rotavirus SA11 and/or human rotavirus RV-5 by 30 to 90% in additive fashion (9, 11). Infectivity blockade in MA104 cell monolayers...
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 ␣21 and ␣41. 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 ␣21 or ␣41 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 ␣21 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 ␣21 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 ␣21 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, ␣21 and ␣41, 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...
Rotavirus replication occurs in vivo in intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins α2β1, α4β1 and αXβ2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed α2β1 and (when tested) αXβ2, whereas the non-permissive K562 cells did not express α2β1, α4β1 or αXβ2. Only RD cells expressed α4β1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface α2 integrin correlated with levels of rotavirus growth. The α2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0n1, and the data best fitted a sigmoidal dose-response curve (r 2 l 1n00, P l 0n005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of α2β1 integrin and is consistent with their expression of αXβ2 and α4β1 integrins.
HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate-labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease.
Group A rotaviruses are major intestinal pathogens that express potential a4b1 and a4b7 integrin ligand sequences Leu-Asp-Val and Leu-Asp-Ile in their outer capsid protein VP7, and Ile-Asp-Ala in their spike protein VP4. Monkey rotavirus SA11 can use recombinant a4b1 as a cellular receptor. In this study a new potential a4b1, a4b7 and a9b1 integrin ligand sequence, Tyr-Gly-Leu, was identified in VP4. It was shown that several human and monkey rotaviruses bound a4b1 and a4b7, but not a9b1. Binding to a4b1 mediated the infectivity and growth of monkey rotaviruses, and binding to a4b7 mediated their infectivity. A porcine rotavirus interacted with a4 integrins at a post-binding stage to facilitate infection. Activation of a4b1 increased rotavirus infectivity. Cellular treatment with peptides containing the a4 integrin ligand sequences Tyr-Gly-Leu and Ile-Asp-Ala eliminated virus binding to a4 integrins and infectivity. In contrast, rotavirus recognition of a4 integrins was unaffected by a peptide containing the sequence Leu-Asp-Val or by a mutation in the VP7 Leu-Asp-Val sequence. VP4 involvement in rotavirus recognition of a4b1 was demonstrated with rotavirus reassortants. Swapping and point mutagenesis of a4 surface loops showed that rotaviruses required the same a4 residues and domains for binding as the natural a4 integrin ligands: mucosal addressin cell adhesion molecule-1, fibronectin and vascular cell adhesion molecule-1. Several rotaviruses are able to use a4b7 and a4b1 for cell binding or entry, through the recognition of the same a4-subunit domains as natural a4 ligands. INTRODUCTIONRotaviruses are a major cause of gastroenteritis through intestinal epithelial cell infection and they can spread extraintestinally Mossel & Ramig, 2003). Many group A rotaviruses bind to the a2b1 integrin via its ligand sequence Asp-Gly-Glu (DGE), which projects from the VP5* subunit of the spike protein VP4. These viruses use the outer capsid protein VP7 to interact with integrins axb2 and avb3 during cell entry Dormitzer et al., 2004;Graham et al., 2003;Guerrero et al., 2000;Hewish et al., 2000). Rotavirus infectivity in intestinal and kidney cell lines is inhibited by at least 65 % with combinations of antibodies or ligand peptides directed to these integrins Graham et al., 2003Graham et al., , 2004Guerrero et al., 2000). Integrins are heterodimeric cell adhesion molecules (Hynes, 2002) that act as receptors for many viruses (Triantafilou et al., 2001). Cellular sialic acids, gangliosides and other carbohydrates are also implicated as rotavirus receptors (Ciarlet et al., 2002; Rolsma et al., 1998). Heat-shock protein 70 has a role in rotavirus cell entry (Lopez & Arias, 2004).Integrins and their natural ligands are essential for epithelial cell adhesion to the extracellular matrix, immune responses and lymphocyte trafficking (Hynes, 2002; Mittelbrunn et al., 2004). The a4 integrin subunit pairs with either b1 or b7 to form part of a subfamily that also includes a9b1 (Hynes, 2002). The a4b1 integrin is expressed on mesench...
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