J Gen Virol 86 (2005), 3397-3408; DOI 10.1099/vir.0.81102-0
© 2005 Society for General Microbiology
Rotaviruses interact with 
4
7 and 41 integrins by binding the same integrin domains as natural ligands
Kate L. Graham1,
Fiona E. Fleming1,
Peter Halasz1,
Marilyn J. Hewish1,
Hadya S. Nagesha1,
,
Ian H. Holmes1,
Yoshikazu Takada2 and
Barbara S. Coulson1
1 Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia
2 The University of California, Davis, UC Davis Medical Center, Research III, Suite 3300, 4645 2nd Avenue, Sacramento, CA 95817, USA
Correspondence
Barbara S. Coulson
barbarac{at}unimelb.edu.au
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ABSTRACT
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Group A rotaviruses are major intestinal pathogens that express potential 
4
1 and 47 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 41 as a cellular receptor. In this study a new potential 41, 47 and 91 integrin ligand sequence, Tyr–Gly–Leu, was identified in VP4. It was shown that several human and monkey rotaviruses bound 41 and 47, but not 91. Binding to 41 mediated the infectivity and growth of monkey rotaviruses, and binding to 47 mediated their infectivity. A porcine rotavirus interacted with 4 integrins at a post-binding stage to facilitate infection. Activation of 41 increased rotavirus infectivity. Cellular treatment with peptides containing the 4 integrin ligand sequences Tyr–Gly–Leu and Ile–Asp–Ala eliminated virus binding to 4 integrins and infectivity. In contrast, rotavirus recognition of 4 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 41 was demonstrated with rotavirus reassortants. Swapping and point mutagenesis of 4 surface loops showed that rotaviruses required the same 4 residues and domains for binding as the natural 4 integrin ligands: mucosal addressin cell adhesion molecule-1, fibronectin and vascular cell adhesion molecule-1. Several rotaviruses are able to use 47 and 41 for cell binding or entry, through the recognition of the same 4-subunit domains as natural 4 ligands.
Present address: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, Ohio State University, Wooster, OH 44691, USA. 
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INTRODUCTION
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Rotaviruses are a major cause of gastroenteritis through intestinal epithelial cell infection and they can spread extraintestinally (Iturriza-Gomara et al., 2002
; Mossel & Ramig, 2003). Many group A rotaviruses bind to the 21 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 x2 and v3 during cell entry (Coulson et al., 1997; 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 (Coulson et al., 1997; Graham et al., 2003, 2004; Guerrero 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 4 integrin subunit pairs with either 1 or 7 to form part of a subfamily that also includes 91 (Hynes, 2002). The 41 integrin is expressed on mesenchymal cells in the intestinal lamina propria. These cells are important for mucosal inflammation and repair (Choy et al., 1990; Pender et al., 2000; Powell et al., 1999). The 4 integrins are highly expressed on B and T lymphocytes, dendritic cells, monocytes and natural killer cells (Hynes, 2002; Pender et al., 2000). Intestinal epithelial cells express 91 (Basora et al., 1998; Yokosaki et al., 1999). The intestinal homing receptor, 47, binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on post-capilliary venules. In contrast, 41 preferentially engages fibronectin and vascular cell adhesion molecule-1 (VCAM-1) to induce intestinal mesenchymal cell migration (Pender et al., 2000) and control B- and T-lymphocyte development, proliferation and survival (Mittelbrunn et al., 2004; Tidswell et al., 1997).
Protection from rotavirus reinfection is primarily mediated by rotavirus-specific intestinal immunoglobulin A (IgA) in animals (Franco & Greenberg, 1999), and correlates with this IgA in humans (Coulson et al., 1992). The presence of rotavirus-specific, antibody-secreting cells in the intestine correlates with their detection in blood (Brown et al., 2000; Yuan et al., 1996). Human rotavirus-specific, antibody-secreting B cells and CD4+ T cells predominantly express 47 (Gonzalez et al., 2003; Rott et al., 1997). In mice, rotavirus-specific B cells require 47 for protective immunity (Williams et al., 1998; Youngman et al., 2002), and memory T cells strongly express 47 (Rose et al., 1998; Rott et al., 1997). Thus, effective rotavirus immune responses depend on MAdCAM-1 recognition by lymphocytes expressing 47.
Potential ligand sequences for 41 and 47 are found in the outer capsid proteins of most rotaviruses. The sequences Ile–Asp–Ala (IDA) and Leu–Asp–Val (LDV) in fibronectin that are important for 41 and 47 recognition (Chan et al., 1992; Komoriya et al., 1991; Ruegg et al., 1992; Sharma et al., 1999; Wayner & Kovach, 1992) are present in VP5* and the disintegrin-like domain of VP7, respectively. This VP7 domain also contains the sequence Leu–Asp–Ile (LDI) that is closely related to LDV (Coulson et al., 1997; Hewish et al., 2000). Simian rotavirus SA11 binds cell surface-expressed 41, facilitating infection and replication (Hewish et al., 2000). Mouse polyomavirus also contains LDV sequence, and uses 41 as an entry factor (Caruso et al., 2003).
Two aims of this study were to determine if human and porcine rotaviruses interact with 41 and to detect any rotavirus recognition of 47. The presence in rotavirus VP5* of the Tyr–Gly–Leu (YGL) sequence used by osteopontin to bind 4 and 9 integrins (Barry et al., 2000; Green et al., 2001; Yokosaki et al., 1999) is reported here for the first time, leading to the hypothesis that this YGL sequence might mediate rotavirus recognition of 4 integrins. As 4 and 9 integrins share ligand specificity, rotavirus usage of 91 was examined. The roles of rotavirus IDA, LDV, LDI and YGL sequences in 4 integrin recognition were studied here for the first time in cells with demonstrable 4 expression.
Integrin subunits combine extracellularly to form a head and two legs. These domains exhibit structural rearrangements between the ‘closed’, low affinity conformation and the ‘open’, high affinity activated state (Hynes, 2002; Xiao et al., 2004). The N-terminal 
440 aa of integrin -subunits contain regions important for ligand binding and consist of seven sequence repeats that are predicted to fold into a -propeller domain in the integrin head (Springer, 1997). Propeller blades are connected by loop structures. Loops that are critical for 4 adhesion to VCAM-1, fibronectin and MAdCAM-1 are grouped in the upper face of the -propeller (Higgins et al., 2000; Irie et al., 1997; Ruiz-Velasco et al., 2000). Another hypothesis tested here was that rotaviruses bind 41 through the recognition of the same 4 sequences as natural 4 ligands.
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METHODS
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Antibodies, proteins and peptides.
Monoclonal antibodies (mAbs) P4C2 (4), 8A2, 4B4 (1) and MOPC21 were obtained as before (Coulson, 1997; Coulson et al., 1997; Hewish et al., 2000). mAbs AK7 (2) and Fib27 (7) were purchased from Becton Dickenson PharMingen, P4G9 (4) from Chemicon and Y9A2 (9) from Serotec. mAb RA3-6B2 (rat anti-mouse B220) was provided by J. Allison, Dept. of Microbiology and Immunology, The University of Melbourne, Australia. BSA and fibronectin were obtained and used as described previously (Graham et al., 2003). Peptides Ser–Val–Val–Tyr–Gly–Leu–Arg (SVVYGLR), Val–Arg–Val–Gly–Leu–Tyr–Ser (VRVGLYS), Ile–Asp–Ala–Pro–Ser (IDAPS), Ile–Glu–Ala–Pro–Ser (IEAPS), Glu–Ile–Leu–Asp–Val–Pro (EILDVP), Glu–Ile–Leu–Glu–Val–Pro (EILEVP), Asp–Gly–Glu–Ala (DGEA) and Gly–His–Arg–Pro (GHRP; 
90 % pure by HPLC) were purchased from Auspep. Peptide SVVYGLR also was provided by A. Albiston, Howard Florey Institute of Experimental Physiology and Medicine, Melbourne, Australia.
Cell lines and viruses.
The generation and maintenance of the cell lines utilized in this study and monitoring of their cell surface integrin expression by flow cytometry were carried out as described previously (Eto et al., 2000; Hewish et al., 2000; Higgins et al., 2000; Irie et al., 1995, 1997; Londrigan et al., 2000). The cell lines utilized were: human K562, Caco-2 and rhabdomyosarcoma (RD), hamster CHO B2 and CHO K1, and monkey MA104 cells; K562 cells transfected with cDNA encoding human integrin subunits 3 (3-K562), 4 (4-K562), 9 (9-K562) and empty vector (PBJ-K562); CHO B2 cells transfected with cDNA encoding human 4 integrin-subunit (CHO B2 4), mutant 4 in which 4-subunit loops were replaced with the corresponding 5 integrin-subunit regions R1 (residues 40–52), R2 (residues 112–131), R3a (residues 151–164), R3b (residues 181–189), R3c (residues 186–191), R4 (residues 237–247) and R5 (residues 282–288) (CHO B2 4 R1, CHO B2 4 R2, CHO B2 4 R3a, CHO B2 4 R3b, CHO B2 4 R3c, CHO B2 4 R4 and CHO B2 4 R5, respectively) and human 4 and 7 (47-CHO B2); CHO K1 cells transfected with cDNA encoding human 4 (CHO K1 4) and 4 with point mutations Y120A/G130A (CHO K1 4 Y120A/G130A), Y187A (CHO K1 4 Y187A) and G190A (CHO K1 4 G190A), and human 4 and 7 (47-CHO K1). The presence of the expected epitopes on recombinant human 4 and 7 expressed on the cell surface was confirmed by flow cytometry using mAbs P4C2 and P4G9 for wild-type and mutant 4, respectively, and mAb Fib27 for 7. The origins of the rotaviruses that were used in this study, and their cultivation in MA104 cells, have been described previously (Graham et al., 2003; Kirkwood et al., 1993).
Virus binding, infectivity and growth assays.
The assays used to determine the titre of infectious rotavirus bound to cells, and measure peptide and mAb inhibition of virus binding were carried out as described previously (Coulson et al., 1997; Graham et al., 2003, 2004; Hewish et al., 2000; Londrigan et al., 2003). Briefly, washed cells were incubated at 37 °C with peptides (0·5 mM) at pH 7·5 for 1 h or mAbs (10 µg ml–1) for 2 h. This antibody concentration was determined by flow cytometry to be the lowest saturating concentration. In the presence of peptide or antibody, cells were cooled on ice for 20 min. Trypsin-activated virus (m.o.i. of 3·5 unless otherwise stated) was cooled to 4 °C and allowed to attach to cells on ice for 1 h. Cell-bound virus titres were determined by indirect immunofluorescent staining of infected MA104 cells, and expressed as focus-forming units per ml (f.f.u. ml–1) (Hewish et al., 2000). The effects of peptides at 0·5 mM (unless otherwise stated), and mAbs on virus infectivity (m.o.i. of 0·02) and growth (m.o.i. of 0·35) were measured by a fluorescence focus reduction-assay (Coulson et al., 1997; Graham et al., 2004; Hewish et al., 2000; Londrigan et al., 2003). For mock infection, cell cultures were inoculated with lysates of confluent, uninfected MA104 cells that had been trypsin-treated and diluted as for the virus infectivity activation. The error bars on the graphs presented represent the standard deviation (SD).
Flow cytometric assay of virus infectivity.
The proportion of PBJ-K562 and 4-K562 cells that were rotavirus-infected was determined by flow cytometry, as these cells showed non-adherent growth. Cells (5x105) that had been infected with trypsin-activated rotaviruses RRV, CRW-8, Wa or RV-5 (m.o.i. of 10) for 16 h were washed once with PBS containing 1 % (v/v) fetal calf serum (PBS/FCS). Cells were permeabilized by methanol fixation for 7 min at –20 °C and washed with PBS/FCS. Cell-associated viral antigen was detected with rabbit antiserum to RRV, using as a negative control normal rabbit serum in which antibodies to rotavirus were present at a titre of <1 : 100 by ELISA (Graham et al., 2003). Cells were further processed for flow cytometry using a two-step stain. This flow cytometric method was validated using rotavirus-infected MA104 cells, by comparison with visual counting of infected cells in fixed monolayers stained by indirect immunofluorescence with the same antiserum (Hewish et al., 2000).
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RESULTS
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Potential ligand sequences for 41, 47 and 91 in rotavirus
The YGL integrin ligand sequence was identified in 164 of 168 (98 %) of VP4 sequences in the Entrez Database (NCBI), in VP5* at aa 448–450. The YGL sequence in osteopontin binds 41 and 91, and antagonizes 47 binding by MAdCAM-1 (Barry et al., 2000; Green et al., 2001; Yokosaki et al., 1999). In 60 of 156 (39 %) of rotaviruses, VP4 contains the IDA sequence in VP5* at aa 538–540. Also, in 272 of 605 (45 %) of rotaviruses, VP7 sequences contain Leu–Asp–Val–Thr (LDVT) at aa 237–240 and 78 % contain Leu–Asp–Ile–Thr (LDIT) at aa 269–272 (Coulson et al., 1997). The sequences present in rotaviruses studied here are indicated in Table 1.
Rotavirus proteins involved in 41 recognition
Rotavirus binding to human 41 was examined using an assay developed previously that detected SA11 binding to 41 on K562 cells, and binding of many rotaviruses to 21 on a range of cell types (Graham et al., 2003, 2004; Hewish et al., 2000; Londrigan et al., 2003). RRV purification did not alter its binding or infectivity using 41 on CHO K1 cells, so clarified virus cell harvests were used as before. Rotaviruses Wa, RV-5, SA11 and RRV that use 21, x2 and v3 for cell binding and entry also bound 41 (Table 1
). Strain K8 that uses 21, x2 and v3, and integrin-independent virus CRW-8 did not bind 41. The rotavirus strain, ST-3, does not use 21, x2 or v3, but did bind 41. Thus, for 5 of 7 rotaviruses, 41 usage correlated with that of other integrins. The two P serotype 1A rotaviruses Wa and F45 differed in their ability to bind 41, showing that the P type did not entirely determine 41 usage. Wa and RV-5 lack IDA, and Wa, RV-5 and ST-3 lack LDVT, indicating that these sequences were not essential for 41 binding. CRW-8 and K8 have YGL and LDIT but did not bind 41. However, all rotaviruses that bound 41 possessed YGL and LDIT.
The identity of the viral gene(s) involved in 41 usage was examined using laboratory-generated reassortants that contained a heterologous VP7 gene segment. Several of these reassortants were used previously to map rotavirus recognition of other integrins (Graham et al., 2003). In three reassortants, the 41-binding phenotype segregated with VP4 (Table 1). A further three reassortants bound 41, consistent with the phenotypes of their parental viruses. Rotavirus usage of 41 did not co-segregate with any other viral gene.
Rotavirus binding to 41 and 47 depended on the ligand-binding domain of 4 and was inhibited by integrin ligand peptides
The ability of rotaviruses to bind recombinant human 47 was tested by comparing virus binding to CHO B2, 4-CHO B2 and 47-CHO B2 cells. Flow cytometric histograms of 4 expression, determined with mAb P4C2, were identical for 4-CHO B2 and 47-CHO B2 cells, so their cell surface levels of 4 were indistinguishable. The relative linear median fluorescence intensity (MFI) of 4 expression was calculated from these histograms, using previously described methodology (Graham et al., 2003), as 3·5 and 3·2, respectively. As expected, only 47-CHO B2 cells expressed the 7-subunit, which was detected with anti-7 antibody Fib27. The MFI of 7 expression on 47-CHO B2 cells was 2·8. CHO B2 cells did not express 4 or 7, as they showed an MFI of 1·0 for these integrin subunits. 4-CHO B2 cells did not express 7 (MFI of 0·8). Similar numbers of 4-K562 cells and K562 cells transfected with 47 bind fibronectin CS-1 and VCAM-1 (Guerrero-Esteo et al., 1998; Munoz et al., 1997; Ruiz-Velasco et al., 2000). Thus, alteration of the 41 activation state on CHO cells by 7 expression is unlikely.
SA11, RRV and Wa bound to human 4 combined with hamster 1 on 4-CHO B2 cells, but bound to a higher level to 47-CHO B2 cells that expressed both human 4 combined with hamster 1, and human 47 (Fig. 1). Thus, these viruses bound to both 47 and 41 on 47-CHO B2 cells. Similar results were obtained using 47-CHO K1 and 4-CHO K1 cells (data not shown). SA11 binding to 4-K562 cells was abolished by cellular treatment with mAb P4C2 (Hewish et al., 2000). This antibody maps to the second and third N-terminal repeats (R2 and R3) of 4 that contain residues critical for ligand binding (Irie et al., 1995, 1997; Kamata et al., 1995). P4C2 also eliminated RRV and Wa binding to 4-K562 cells, as virus titres bound in the presence of this antibody were indistinguishable from those bound to PBJ-K562 and 3-K562 cells (Fig. 2). P4C2 similarly abrogated rotavirus binding to 47. In contrast, the function-blocking mAb Fib27 that maps to aa 176–237 of 7 (Tidswell et al., 1997) had no effect on virus binding to 47. Similarly, function-blocking anti-1 antibody 4B4 that inhibited SA11 binding to MA104 cells (Coulson, 1997) did not affect RRV binding to 41. CRW-8 binding to CHO and K562 cells was independent of 41 or 47 and unaffected by the anti-4 or -7 mAbs.
Peptides containing the integrin ligand sequences present in rotaviruses were used to further define the roles of these sequences in 4 binding. Peptides used as negative controls were the same as those included in previous studies of integrin recognition of cellular ligands. Peptides inhibitory to rotavirus binding were SVVYGLR that binds 41 and 47, derived from osteopontin (Barry et al., 2000; Green et al., 2001), and IDAPS, constituting a region of fibronectin H1 that has an indirect role in 41 binding. IDAPS peptide blockade of 41 binding to fibronectin has been proposed to result from mimicry of the EILDVP sequence in fibronectin that directly binds 41 and 47 (Chan et al., 1992; Komoriya et al., 1991; Ruegg et al., 1992; Sharma et al., 1999; Wayner & Kovach, 1992). SVVYGLR and IDAPS eliminated SA11, RRV and Wa binding to recombinant, cell surface-expressed 41 and 47 (Fig. 2). However, peptide EILDVP, and control peptides VRVGLYS, IEAPS and EILEVP did not affect binding of these viruses to 4 integrins. A low level of SA11 inhibition was seen with VRVGLYS. None of the peptides altered the background level of binding of CRW-8 to the transfected cells.
Infectivity and growth of some rotaviruses were mediated through 41 and 47
SA11 binding to 41 resulted in increased SA11 yield (Hewish et al., 2000). The abilities of other rotaviruses to infect and replicate via 41 were determined. In the presence of 41, the number of K562 cells infected by RRV and CRW-8 (detected by flow cytometry) increased from a mean±SD of 45·3±1·9 to 56·9±2·0 % and from 0·1±0·0 to 24·7±0·4 %, respectively (Fig. 3a). RRV and CRW-8 yields increased four- to sixfold due to 4 expression (Fig. 3b). The negligible proportion of 4-K562 cells infected by Wa (0·2±0·1 %) was similar to that in K562 cells (0·1±0·1 %) and mock-infected cells (0·2±0·1 %), and no infectious Wa or RV-5 was produced by PBJ-K562, 3-K562 or 4-K562 cells at 24–72 h post-infection (p.i.) (data not shown). RRV and CRW-8 growth in 4-K562 cells was inhibited by SVVYGLR and IDAPS (Fig. 3c), the same peptides that abolished virus binding to 4 integrins (Fig. 2). Thus, although SA11, RRV, Wa and RV-5 all bound 41, only SA11 and RRV were able to utilize this binding for infection and virus growth in K562 cells. Conversely, CRW-8 interacted with 41 to greatly increase its infectivity, but did not use this integrin for initial cell binding.
Rotavirus usage of naturally expressed 41 on RD cells (Londrigan et al., 2000) was studied. P4C2 and fibronectin showed a dose-dependent blockade of RRV infectivity in RD cells, to 26±7·9 % at 10 µg ml–1 and 35±3·9 % at 160 µg ml–1, respectively. P4C2 did not affect RRV (data not shown) or SA11 (Coulson et al., 1997) infectivity in 4 integrin-negative MA104 cells.
CHO B2 cells lack 5 integrin expression, so they lost adhesion during prolonged incubation at 37 °C (Schreiner et al., 1989). Therefore, rotavirus infectivity mediated by 47 was determined using 47-CHO K1 cells. The mean±SD of the respective SA11 and RRV titres in f.f.u. ml–1 produced at an m.o.i. of 40 in 47-CHO K1 cells (5800±750 and 6500±1000) were significantly higher than those in CHO K1 cells (2400±750 and 3300±850) at 16 h p.i. (t-test, P<0·0001). Wa and CRW-8 did not infect either cell line. Thus, binding of SA11 and RRV, but not Wa, to 47 resulted in increased infectivity in CHO cells.
Rotaviruses did not utilize 91 for cell attachment or entry
SA11, RRV and CRW-8 did not use recombinant 91 for K562 cell infection (Fig. 3b) or replication (Fig. 4a, lower panel). SA11 (Fig. 4a, upper panel) and Wa (data not shown) did not bind 91. Caco-2 cell expression of 9 was detected by flow cytometry with anti-9 mAb Y9A2 (data not shown). This antibody did not affect SA11, RRV and Wa infection of Caco-2 cells, and peptide SVVYGLR did not affect SA11 infection of Caco-2 cells (Fig. 4b). Thus, 91 was not a rotavirus receptor or entry co-factor.
Activation of 1 increased SA11 growth in 4-K562 cells
Activating antibodies induce integrins into high affinity states for ligand binding. Eosinophil resistance to 41-mediated detachment from VCAM-1 and MAdCAM-1 is increased by activating anti-1 antibody 8A2 (Sriramarao et al., 2000). To determine if 1 activation affects rotavirus–41 interactions, SA11 replication was measured in 4-K562 cells that had been treated with 8A2 at the concentration (0·1–0·2 µg ml–1) previously shown to increase K562 cell binding to fibronectin (Faull et al., 1996) and SA11 binding to 21 (Graham et al., 2003). Activation of 1 increased SA11 yield from 4-K562 cells by 50–70 % at 1 and 24 h p.i. (Fig. 5a). Thus, rotavirus infectivity was enhanced by 41 activation.
Mutation of LDVT in VP7 of SA11 did not affect 41 recognition
The minimal active site of the dominant 41-binding region in fibronectin is LDV, which is totally dependent on its Asp residue (Komoriya et al., 1991). The role of VP7 LDVT sequence in rotavirus replication was directly analysed using an SA11 variant, V-SA11-A10II, which has a single mutation in the Asp residue of the LDVT sequence (D238N) (Lazdins et al., 1995). Similarly to SA11, V-SA11-A10II replicated to a higher titre in 4-K562 and 2-K562 cells than in control 3-K562 cells, so VP7 LDVT was not required for SA11 usage of 41 or 21 (Fig. 5b). V-SA11-A10II grew to a 10-fold higher titre than SA11 in these K562 cells. This might be directly related to the LNVT mutation, but the possibility that other viral genes are involved cannot be ruled out.
Domains and sequence of the 4-subunit required for rotavirus binding
The 4 domains crucial for rotavirus binding were located using swapping mutagenesis. Predicted protruding loops at or near 41 sites necessary for natural ligand binding were replaced with corresponding 5 regions (Higgins et al., 2000; Irie et al., 1997). Rotaviruses do not bind 51. Therefore, if the swapped 4 region is necessary for virus binding, swapping to the 5 region will prevent virus binding to 4. The region swapping will affect virus binding only if the exchanged regions are sufficiently distinct in sequence. The R3b and R3c domains are conservative in sequence between 4 and 5. The only point mutations in the putative ligand-binding region of 4 (aa 108–268) of 61 tested that abolish natural 4 ligand binding are non-conservative and located in R2 (Y120/G130), R3b (Y187) and R3c (G190) (Higgins et al., 2000; Irie et al., 1995, 1997). As shown in Fig. 6, 4 was detected at similar levels on all CHO cell lines transfected to express these swapping and point mutations in 4, using anti-4 antibody P4G9 that maps to an epitope outside the swapped domains (Irie et al., 1997; Kamata et al., 1995). Binding of RRV, SA11 and Wa to 41 on CHO B2 cells was abolished by swapping R2 or R4, but was unaffected by swapping R1, R3a, R3b, R3c or R5 (Fig. 7). The point mutations eliminated Wa, RRV and SA11 binding to 41. CRW-8 did not bind 41 on any cell line tested, showing the specificity of the Wa, SA11 and RRV binding (Fig. 7). CS-1 peptide, VCAM-1 and MAdCAM-1 also required 4 R2 and R4, but not R1, R3a, R3b, R3c or R5 for binding (Higgins et al., 2000; Irie et al., 1997). Thus, the same 4 regions and amino acids are critical for 4 binding by both rotaviruses and these natural 4 ligands.
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DISCUSSION
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In this study, the repertoire of integrins used by rotaviruses for cell-binding and post-binding interactions was extended to include 47, an intestinal homing receptor crucial for protective immune responses. It was shown for the first time that human rotaviruses bind 41 and 47, and rotaviruses require the same 4 residues for binding as natural 4 ligands. Three modes of rotavirus interactions with 4 integrins were identified. Firstly, SA11 (and RRV) uses 41 and 47 as receptors, resulting in K562 and CHO K1 cell infection. Secondly, Wa and RV-5 bind 41 but fail to infect K562 cells expressing 41. Thirdly, CRW-8 infectivity in K562 cells is greatly increased by 41 expression, but CRW-8 appears to use 41 as a co-entry factor rather than a receptor. The latter two modes have not been demonstrated previously.
A further novel finding is the likely involvement of the conserved VP5* YGL sequence in 4 recognition by rotaviruses. The peptide SVVYGLR inhibited 41 and 47 binding by rotaviruses and virus infectivity, and 41 binding by rotaviruses segregated with VP4 in reassortants. It is proposed that VP4 binds 41 using YGL. In the X-ray crystallographic structure of RRV VP5* containing aa 252–523 (VP5CT), most of the Tyr residue in the YGL sequence is exposed on the surface (Dormitzer et al., 2004). The shorter sequence SVVYG is sufficient to mediate adhesion to 4 integrins (Gao et al., 2004). Possibly, exposure of the Tyr and Gly residues in YGL may be sufficient for binding. The YGL sequence might become accessible through opening of a possible diglycine hinge in overlying -sheets (Dormitzer et al., 2004).
The VP5* IDA sequence was not necessary for 4 integrin binding by rotaviruses, consistent with the IDA location in a predicted heptad repeat region that may be inaccessible (Bremont et al., 1992; Lopez et al., 1991). Peptide IDAPS elimination of rotavirus binding to 4 integrins probably occurred via non-IDA viral sequence, similarly to IDAPS inhibition of fibronectin binding to 4 through EILDVP mimicry (Sharma et al., 1999). SVVYGLR and EILDVP peptides compete for 41 binding, and have similar affinities for 41 (Barry et al., 2000). As SVVYGLR, IDAPS and EILDVP peptides inhibit ligand binding through related 4 regions, the differential effects of these peptides on rotavirus binding and infection probably relate to sequence differences in parts of the peptides with portions of the 4-binding regions in rotavirus. The VP7 LDV sequence was not involved in 4 integrin binding, as the presence of LDVT, LDVT mutation and EILDVP blockade did not affect 4 interactions with rotaviruses. This lack of inhibition by EILDVP also suggests that the VP7 LDI is not necessary for 4 recognition by rotaviruses.
Anti-VP5* mAb 2G4, and anti-VP7 antibodies RV-4 : 2 and F45 : 2, inhibited rotavirus–41 binding to a titre similar to their neutralization titres. Acute- and convalescent-phase sera from children with gastroenteritis also specifically inhibited Wa binding to 41 (K. L. Graham, F. E. Fleming, P. Halasz, Y. Takada and B. S. Coulson, unpublished results). These findings support the proposed roles for VP4 and VP7 in 41 recognition by rotaviruses.
Rotavirus usage of 4 integrins but not 91 was unexpected, as SVVYGLR is a common ligand for these integrins. Mutation of the SVVYGLR peptide showed that Leu and Tyr are important for 4 binding (Green et al., 2001), whereas the Tyr appears to be critical for 91 binding (Yokosaki et al., 1999). The first Val has a role in 4 binding, and mutation of the Arg increased activity against 41 but reduced activity against 47 (Green et al., 2001). Non-conservative amino acid changes for the Val and Arg adjacent to YGL in VP5* could prevent 91 binding by rotaviruses.
It is at present unclear how rotavirus interactions with 4 fit with current models of rotavirus cell entry. SA11 and RRV binding to 41 and 47 may facilitate virus cell entry by endocytosis or direct membrane penetration (Lopez & Arias, 2004), as proposed previously for rotavirus usage of other integrins (Graham et al., 2003). Human rotavirus binding to 4 might affect cellular functions that relate to antirotaviral responses, without facilitating infection. CRW-8 cell binding is independent of cellular protein synthesis (Jolly et al., 2001), and OSU, another porcine rotavirus of the same P serotype as CRW-8, binds ganglioside GM3 (Rolsma et al., 1998). Therefore, CRW-8 may bind GM3, facilitating usage of 4 integrins in a co-receptor role that results in cell entry. The identification of several possible modes of rotavirus interactions with 4 in this study will allow further dissection of the mechanisms by which different rotaviruses utilize these integrins.
The adjacent R2, R3b, R3c and R4 loops of 4 in the upper face of the predicted -propeller structure of the integrin head were critical for rotavirus 4 recognition. However, loops R1 and R5 that are located distal from R2, R3b R3c and R4 on the upper or lower face, respectively (Higgins et al., 2000; Irie et al., 1997; Springer, 1997), were not involved in rotavirus 4 binding. The 4 loops required for binding by rotaviruses, MAdCAM-1, fibronectin and VCAM-1 are, therefore, identical (Higgins et al., 2000; Irie et al., 1995, 1997). Judging from their predicted positions, these loops are highly exposed on the head of activated 4 and ideally placed to mediate binding to rotaviruses and natural ligands. The increased rotavirus infectivity after 41 activation supports this conclusion.
This binding of rotaviruses to 4 integrins through the same 4 domains as natural ligands strongly suggests that virus 4 binding is likely to affect the functions of these competing ligands. The blockade of rotavirus 4 binding by SVVYGLR reinforces this conclusion, as SVVYGLR completely inhibits 47 binding to MAdCAM-1 and 41 binding to fibronectin CS-1, and partially inhibits 41 binding to VCAM-1 (Green et al., 2001). Rotavirus 47 binding might affect immune responses dependent on 47 recognition of MAdCAM-1 for intestinal homing, and modulate the protection afforded against reinfection by rotavirus-specific 47hi B and T cells (Gonzalez et al., 2003; Rose et al., 1998; Rott et al., 1997; Williams et al., 1998; Youngman et al., 2002). Potential immune modulation by rotavirus–integrin interactions is a topic for further study.
Lamina propria mesenchymal cells are the only resident gut cell known to express 4 (Choy et al., 1990; Pender et al., 2000). Possibly, 41 on these cells may bind rotaviruses. Rotavirus disease may be associated with viraemia (Blutt et al., 2003), so virus in the circulation could contact immune cells expressing 4. Rotavirus recognition of activated 41 might facilitate virus carriage and infection. Lymphoid cells mediate rotavirus escape from the gut (Mossel & Ramig, 2003), which might involve virus 4 integrin binding. Rotavirus infection has been associated with encephalitis (Iturriza-Gomara et al., 2002) and pancreatic islet autoantibody responses (Honeyman et al., 2000). Rotavirus 47 binding might disseminate virus within the intestine and to the pancreas and brain where MAdCAM-1 is also expressed. These issues are important subjects for continuing research.
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INTRODUCTION
