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2
1 integrin on the cell surface and competes with virus for cell binding and infectivity
1 Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia
2 The University of California, Davis, UC Davis Medical Center, 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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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2
1 integrin, and the processes of rotavirus cell attachment and entry appear to be multifactorial. The VP5* subunit of the rotavirus spike protein VP4 contains the 21 ligand sequence Asp–Gly–Glu at residues 308–310. Binding to 21 and infectivity of monkey rotavirus strain RRV and human rotavirus strain Wa, but not porcine rotavirus strain CRW-8, are inhibited by peptides containing Asp–Gly–Glu. Asp308 and Gly309 are necessary for the binding of RRV VP5* (aa 248–474) to expressed I domain of the 2 integrin subunit. Here, the ability of RRV VP5* to bind cells and affect rotavirus–integrin interactions was determined. Interestingly, VP5* bound to cells at 4 and 37 °C, both via 21 and independently of this integrin. Prior VP5* binding at 37 °C eliminated RRV binding to cellular 21 and reduced RRV and Wa infectivity in MA104 cells by 38–46 %. VP5* binding did not affect the infectivity of CRW-8. VP5* binding at 4 °C did not affect permissive-cell infection by RRV, indicating an energy requirement for VP5* competition with virus for infectivity. Mutagenesis of VP5* Asp308 and Gly309 eliminated VP5* binding to 21 and the VP5* inhibition of rotavirus cell binding and infection, but not 21-independent cell binding by VP5*. These studies show for the first time that expressed VP5* binds cell-surface 21 using Asp308 and Gly309 and inhibits the infection of homologous and heterologous rotaviruses that use 21 as a receptor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In group A rotaviruses, VP4 and VP7 dictate the P and G serotypes, respectively. VP4 is the major viral cell-attachment protein, whereas VP7 has a lesser role (Bass et al., 1991; Crawford et al., 1994; Kirkwood et al., 1998; Ludert et al., 1996). Proteolytic cleavage of VP4 by trypsin at residue 247 results in greatly increased infectivity and produces two subunits, VP5* (aa 248–776) and VP8* (aa 1–247) that remain associated with the virion (Espejo et al., 1981; Estes et al., 1981; Gilbert & Greenberg, 1998). Trypsin treatment does not appear to alter virus–cell binding, but increases the rate of cell entry by an unknown mechanism (Clark et al., 1981; Kaljot et al., 1988). However, it is known that trypsin-treated, but not untreated, virions can induce cellular fusion from without (Falconer et al., 1995). Also, expressed VP5* permeabilizes membranes (Denisova et al., 1999; Dowling et al., 2000; Golantsova et al., 2004). Trypsin cleavage of VP4 results in dimeric VP4 spikes with a well-ordered structure (Crawford et al., 2001), suggesting the induction of a conformational change in VP4. Crystal structures of most of the portions of VP8* and VP5* that project from the virion show that VP4 also undergoes a second rearrangement that may facilitate VP4–membrane interactions and results in a stable trimer (Dormitzer et al., 2002, 2004). At high pH, VP4 spikes on the virion undergo conformational change to a trilobed structure (Pesavento et al., 2005).
Rotaviruses recognize several cell-surface molecules, and the processes of rotavirus cell attachment and entry appear to be multifactorial. Binding of a minority of animal rotaviruses to terminal sialic acids has been demonstrated, although this interaction is not essential (Ludert et al., 2002; Mendez et al., 1993). Terminal sialic acid usage relates to P serotype (Ciarlet et al., 2002b), and VP8* of the neuraminidase-sensitive rotavirus strain RRV contains a sialoside-binding region involved in cell binding and haemagglutination (Dormitzer et al., 2002; Fiore et al., 1991). Other rotaviruses, including some human strains, may recognize subterminal sialic acids that are not removed by neuraminidase treatment, or other sugars, on glycoproteins or glycolipids including gangliosides (Delorme et al., 2001; Fukudome et al., 1989; Guo et al., 1999; Jolly et al., 2000, 2001a; Liakatos et al., 2006; Rolsma et al., 1998).
Several human and monkey rotaviruses bind recombinant, cell-surface-expressed 41 and 47 integrins and require the same 4 subunit regions for binding as natural 4 ligands. Rotaviruses may recognize 4 integrins on cells of the immune system, possibly facilitating virus spread or host immune response modulation (Graham et al., 2005; Halasz et al., 2005; Hewish et al., 2000). The heat-shock cognate protein 70 (Hsc70) has also been proposed to form a component of a rotavirus receptor complex that is recognized through aa 642–659 of VP5* (Zárate et al., 2003).
Infection of permissive cells by many human and animal rotaviruses depends, to a significant extent, on VP4 recognition of 21 integrin and VP7 interactions with integrins x2 and v3 (Ciarlet et al., 2002a; Coulson et al., 1997; Graham et al., 2003, 2004; Guerrero et al., 2000). These rotaviruses are classified as integrin-using strains and include monkey virus strains RRV and SA11 and human strain Wa. Integrin usage relates to P serotype independently of terminal sialic acid usage (Graham et al., 2003). Almost all group A rotaviruses have the Asp–Gly–Glu (DGE) sequence in VP5* at aa 308–310, a motif that has been implicated in 21 recognition by type I collagen, and the Gly–Pro–Arg (GPR) sequence in VP7 that is a ligand in fibrinogen for x2 (Coulson et al., 1997). Monomeric and polymeric peptides containing the DGE and/or GPR sequences inhibit the infectivity of integrin-using rotaviruses by 30–90 % (Coulson et al., 1997; Graham et al., 2004; Zárate et al., 2000a). Integrin-using rotaviruses have been reported to use x2 and v3 at a post-binding stage to facilitate infection (Graham et al., 2003; Guerrero et al., 2000).
Most evidence indicates that integrin-using human and animal rotaviruses bind to cell-surface 21. Infectious SA11, RRV and Wa binding to recombinant 21 on the K562 cell surface was specifically inhibited by DG-containing peptides and a function-blocking antibody to the 2 I domain (Graham et al., 2003, 2004; Hewish et al., 2000). SA11 and RRV precipitated two cell-surface proteins with characteristics of 2 and 1 integrin subunits and bound recombinant human 21 on Chinese hamster ovary cells to a greater extent than human 2 combined with hamster 1. This binding was inhibited by antibody to the 2 I domain, but not anti-2 antibodies that mapped outside the I domain, and was eliminated by I domain deletion (Londrigan et al., 2003). VP5* (aa 248–474, representing the N-terminal half of VP5*), expressed as a glutathione S-transferase (GST) fusion protein, retained the ability to bind a conformation-dependent neutralizing antibody and bound expressed 2 I domain. This I domain binding depended on the presence of the D308 and G309 residues in the DGE sequence of VP5*, as alanine mutagenesis of these residues in VP5* did not affect neutralizing-antibody recognition, but did abolish VP5* binding to the 2 I domain (Graham et al., 2003). In contrast to the above findings, binding of naturally occurring, integrin-using rotaviruses to cellular 21 was not detected by two other groups (Ciarlet et al., 2002a; Zárate et al., 2000a, b). One reason for this was their use of normally adherent MA104 cells as cell suspensions during virus–cell binding assays (Graham et al., 2003). However, recent studies by one of these groups using adherent MA104 cells still failed to demonstrate 21 binding by RRV (Zárate et al., 2003, 2004).
To examine the mechanism of rotavirus binding to cellular 21 further and resolve this difference, we aimed here to analyse directly the importance of VP5* (aa 248–474) containing the DGE sequence in rotavirus cell binding and infection mediated by 21. These studies provide the first direct experimental evidence that purified recombinant VP5* binds cell-surface 21 using DGE and inhibits the infection of homologous and heterologous rotaviruses that use 21 as a receptor.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2 (2-K562), 3 (3-K562) and 4 (4-K562) used in this study have been described previously (Coulson et al., 1997; Graham et al., 2003; Hewish et al., 2000). By flow cytometry, MA104 cells express moderate levels of surface 21 (Coulson et al., 1997; Londrigan et al., 2000). Monitoring of the surface expression of 21, 31 and 41 on K562 cell lines was carried out by flow cytometry as described previously (Graham et al., 2005; Hewish et al., 2000). The origins, cultivation in MA104 cells and characterization of Rhesus monkey rotavirus P5B[3], G3 strain RRV; human rotavirus P1A[8], G1 strain Wa and porcine rotavirus P9[7], G3 strain CRW-8 have been described previously (Coulson & Kirkwood, 1991; Coulson et al., 1985, 1986). Rabbit antiserum to purified GST and RRV were each produced and obtained as described previously (Londrigan et al., 2003; Warner et al., 2001).
Production and characterization of purified recombinant VP5* proteins.
The RRV VP4 plasmid pBS/VP4 was provided by Dr E. Mackow (Department of Medicine and Department of Molecular Genetics and Microbiology, Stony Brook University, NY, USA). Soluble RRV VP5* containing aa 248–474 and the D308A, G309A mutant of this VP5* generated by site-directed mutagenesis were produced as purified GST fusion proteins GST–VP5* and GST–VP5*D308A/G309A, respectively, by Escherichia coli expression as described previously (Graham et al., 2003). Most of the GST–VP5* and GST–VP5*D308A/G309A protein produced was insoluble, as reported previously (Dormitzer et al., 2004), but under the expression conditions used, a fraction that co-purified with the heat-shock bacterial chaperonin GroEL was soluble, as determined previously (Graham et al., 2003). By enzyme immunoassay (EIA), the soluble GST–VP5* and GST–VP5*D308A/G309A produced for the studies here were bound by anti-VP5* monoclonal antibody 2G4 (indicating preservation of this conformational epitope), and GST–VP5* but not GST–VP5*D308A/G309A bound to the 2 integrin I domain fusion protein, as described previously (Graham et al., 2003). The soluble protein obtained therefore was functional for 2G4 recognition and 2 integrin I domain binding. Plasmid insert identity was verified by DNA sequencing. Fusion protein identity was confirmed by Western blotting using anti-GST monoclonal antibody CH-1, as described previously (Graham et al., 2003).
Flow cytometric assay of recombinant VP5* binding to cells.
The ability of GST–VP5*, GST–VP5*D308A/G309A and GST to bind PBJ-K562, 2-K562, 3-K562 and 4-K562 cells was determined by flow cytometry as described previously (Warner et al., 2001). In brief, equimolar amounts of GST–VP5*, GST–VP5*D308A/G309A or GST (0·1–10 µg) were incubated with 5x105 washed cells for 45 min at 4 °C unless stated otherwise. Cell numbers were quantified carefully to ensure that results were comparable between cell lines. All the following steps were carried out at 4 °C. Cell-bound protein was detected with rabbit antiserum to GST diluted 1 : 500. Similarly diluted, normal rabbit serum negative for rotavirus antibodies by EIA and with a neutralization titre against SA11 of <1 : 100 was used as a negative control. Bound rabbit antibodies were detected using fluorescein isothiocyanate-conjugated sheep anti-rabbit F(Ab')2 (Chemicon) and cells were analysed by flow cytometry, as described previously (Graham et al., 2003). The relative level of recombinant protein bound to cells was quantified by calculation of the relative linear median fluorescence intensity (RLMFI) from the flow cytometric histograms, as described previously (Graham et al., 2003). An RLMFI value of 
1·20 was considered to indicate cell binding by the recombinant protein, as has been determined previously for monoclonal antibody binding (Graham et al., 2003).
Indirect immunofluorescence assays of virus–cell binding and infectivity and recombinant VP5* inhibition of virus–cell binding and infectivity.
Measurement of the binding of infectious rotavirus to cells was determined using 5x105 cells and infectivity assays were carried out using 1x104 cells, as described previously (Coulson et al., 1997; Graham et al., 2003; Hewish et al., 2000; Londrigan et al., 2000). Assays were carried out using clarified virus–cell harvests at an m.o.i. of 3·5 (virus–cell binding) or 0·02 (infectivity). The determination of these optimum m.o.i. values, and demonstration that the titres of purified virus and clarified virus–cell harvests bound to cellular integrins are indistinguishable, have been described previously (Coulson et al., 1997; Graham et al., 2003, 2005). Titres are given as fluorescent cell-forming units (f.f.u.) ml–1.
Briefly, for binding assays, trypsin-activated virus was adsorbed to cells at 4 °C for 1 h. Bound virus was harvested by two cycles of freezing and thawing in the presence of 1 µg porcine trypsin ml–1 and activated with 10 µg trypsin ml–1 at 37 °C for 10–20 min, depending on the virus strain. In conjunction with vigorous vortex mixing, these trypsin digestions reduced levels of virion-associated protein and virion aggregation. Cell-bound virus titres were determined by incubation of MA104 cells with activated virus at 37 °C for 1 h, followed by indirect immunofluorescent staining of infected cells after 15 h. In experiments analysing recombinant protein inhibition of virus–cell binding and infectivity, cells were treated with GST–VP5*, GST–VP5*D308A/G309A or GST at 37 °C for 1 h prior to virus addition, unless otherwise stated. Assays were then completed as described above. None of the proteins caused aggregation of PBJ-K562 or integrin-transfected K562 cells. Cell viability (measured by trypan blue exclusion) and the microscopic appearance of MA104 and K562 cell lines were unaltered by protein treatment.
On graphs, results were expressed as a percentage of the virus titre in the absence of any treatment and given as mean±SD of at least three experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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21-dependent and -independent mechanisms2 integrin I domain protein (Graham et al., 2003). The ability of GST–VP5* to bind cell-surface-expressed 21, 31 or 41 was examined at 4 and 37 °C by flow cytometry, using K562 cells transfected with the empty vector (PBJ-K562) or expressing recombinant human 21 (2-K562), 31 (3-K562) or 41 (4-K562). The only endogenous 1 integrin expressed by K562 cells is 51. GST–VP5* bound to PBJ-K562, 2-K562, 3-K562 and 4-K562 cell lines at both 4 °C (Fig. 1a) and 37 °C (Fig. 1b). However, 2-K562 cells supported substantially more GST–VP5* binding than PBJ-K562, 3-K562 and 4-K562 cells at both 4 and 37 °C (Fig. 1a–d). GST–VP5* binding to 2-K562 and 3-K562 cells was concentration-dependent over a 100-fold range (0·1–10 µg GST–VP5*) and was saturated at 5 µg GST–VP5* per 5x105 cells (Fig. 1c and d). RLMFI values calculated from the experiments using GST–VP5* indicated that 2-K562 cells supported 1·3–2·2-fold more GST–VP5* binding than PBJ-K562, 3-K562 and 4-K562 cells (Table 1). Overall, at 4 and 37 °C, GST–VP5* bound to K562 cells using 21 and also via an additional mechanism that was independent of 21, 31 or 41.
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212 integrin I domain protein has been demonstrated to depend on the presence of D308 and/or G309 in VP5*, as RRV GST–VP5*D308A/G309A did not bind the 2 I domain (Graham et al., 2003). The ability of RRV GST–VP5*D308A/G309A to bind K562 cells by 21-dependent and 21-independent means was examined. As shown in Fig. 2 and Table 1
, GST–VP5*D308A/G309A binding to 2-K562 cells was detected (RLMFI=1·4) at a lower level than GST–VP5* binding to 2-K562 cells (RLMFI=3·1). GST–VP5*D308A/G309A and GST–VP5* bound 3-K562 cells indistinguishably, with identical RLMFI values of 1·4, at levels indistinguishable from VP5*D308A/G309A binding to 2-K562 cells (RLMFI=1·4). These results showed that mutation of the DG sequence in VP5* abolished VP5* binding to cell-surface-expressed 21, but that VP5* binding to cells independently of 21 remained detectable.
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21 prevents RRV binding21 was examined (Fig. 3). In the absence of cellular treatment with recombinant protein, the RRV titre bound to 2-K562 cells was increased 1·9-fold over that bound to PBJ-K562 and 3-K562 cells, as shown previously (Graham et al., 2003). Cellular treatment with RRV GST–VP5* at 37 °C eliminated infectious RRV binding to 21 on 2-K562 cells, as the RRV titre bound was indistinguishable from that bound to PBJ-K562 and 3-K562 cells. However, RRV GST–VP5*D308A/G309A and GST had no effect on the titre of RRV bound to 21 on 2-K562 cells and did not alter the background level of RRV binding to PBJ-K562 and 3-K562 cells. Thus, RRV binding to cellular 21 was prevented by prior cellular treatment with homologous GST–VP5* at 37 °C and this inhibition was completely dependent on VP5* D308 and/or G309.
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21 inhibits RRV and Wa infection of MA104 cells21 on K562 cells and so prevent RRV binding to the same integrin, the abilities of these proteins to bind permissive MA104 cells and compete with rotavirus infectivity were examined. Both GST–VP5* and GST–VP5*D308A/G309A bound to MA104 cells, showing RLMFI values of 5·2±0·5 and 4·0±0·4, respectively. These RLMFI values are consistent with the results described above, which demonstrated that GST–VP5* binds K562 cells both via 21 and independently of this integrin, whereas GST–VP5*D308A/G309A binds K562 cells independently of 21 only. The higher RLMFI value shown by GST–VP5* probably reflects its additional ability to bind 21. Application of RRV GST–VP5* to MA104 cells at 4 °C had no effect on the infectivity of RRV added subsequently (Fig. 4a). However, cellular treatment with RRV GST–VP5* at 37 °C inhibited RRV infectivity in a dose-dependent fashion with a mean±SD of 38±4 % at 50 µg ml–1. This infectivity blockade was abrogated by the D308A and G309A mutations in VP5*, as RRV GST–VP5*D308A/G309A did not inhibit RRV infectivity at any concentration tested (Fig. 4b). Importantly, RRV GST–VP5* similarly inhibited infection by the heterologous human rotavirus strain Wa, with a mean of 46±3 % at 50 µg ml–1, whereas RRV GST–VP5*D308A/G309A did not inhibit Wa infectivity (Fig. 4c). Neither RRV GST–VP5* nor RRV GST–VP5*D308A/G309A had any effect on infection by CRW-8 rotavirus (Fig. 4d), which does not use 21 for cell binding or growth (Graham et al., 2003, 2004; Hewish et al., 2000). Thus, RRV GST–VP5* bound to cells at 37 °C but not at 4 °C inhibited RRV and Wa, but not CRW-8, rotavirus infectivity in MA104 cells. This blockade required the presence of VP5* D308 and/or G309 and so was dependent on VP5* binding to 21.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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21 integrin were elucidated further. The direct relevance to rotavirus cell attachment and infectivity of the previously described binding of RRV spike protein VP5* to 2 subunit I domain protein (Graham et al., 2003) was established. It was shown that binding of cellular 21 by infectious rotavirus was inhibited by VP5* (aa 248–474), which bound to cells in a DGE-dependent manner. This cell-bound VP5* resulted in a reduction in infectivity by both homologous and heterologous rotaviruses that use 21 as a cellular receptor.
Rotavirus VP5* binds cellular 21 via D308 and G309 and competes with infectious virus for cell binding and infectivity
RRV VP5* bound K562 cells at 4 and 37 °C, both via 21 and independently of 21, 31 and 41 (Fig. 1). These data extend previous findings with a similar expressed GST–VP5* construct that bound MA104 cells at 4 °C, as binding of this construct was partially inhibited by an anti-2 antibody (Zárate et al., 2003). The requirement for the DGE sequence to mediate VP5* binding to cellular 21 was demonstrated by the inability of GST–VP5*D308A/G309A to bind 21 (Fig. 2). As GST–VP5*D308A/G309A did show 21-independent binding to K562 cells (Fig. 2), mutation of the DGE sequence did not affect 21-independent binding functions. This demonstrated the specificity of the effect of DGE mutation in RRV VP5* on 21 binding.
RRV VP5* (aa 248–474) permeabilizes membranes and directs the formation of transient size-selective pores without membrane lysis. These functions require the hydrophobic and basic domains, but are independent of 21 recognition and have been proposed to mediate rotavirus entry into cells (Denisova et al., 1999; Dowling et al., 2000; Golantsova et al., 2004). The 21-independent cell binding by GST–VP5* and GST–VP5*D308A/G309A (Figs 1 and 2) may have been mediated by these domains. Other VP5* domains may also be responsible for integrin-independent cell binding, such as the MA104 cell-binding domains identified in CRW-8 VP5* using a gene-targeted phage display library (Jolly et al., 2001b). Hsc70 interactions could not account for the integrin-independent binding of VP5*, as the VP5* amino-terminal region that binds Hsc70 was not present in the VP5* used in our studies.
GST–VP5* almost completely blocked binding of RRV to 21 and dose-dependently inhibited MA104 cell infection by RRV and Wa but not CRW-8. These inhibitory functions of VP5* depended on the presence of the DGE sequence and so were specific for 21. The reduction of heterologous rotavirus infection by RRV GST–VP5* depended on the ability of the infecting virus to utilize 21, rather than the terminal sialic acid dependence or VP7 serotype of the virus (Ciarlet et al., 2002b; Nagesha & Holmes, 1991). Approximately 40 % of rotaviruses in the pool failed to enter cells productively when competed with VP5*. This is similar to the levels of blockade of rotavirus cell binding and infectivity by antibodies to 2 and monomeric DGEA peptides (Graham et al., 2004) and is consistent with VP5* blockade of 21 binding being a major mechanism by which VP5* inhibits virus–cell interactions.
The blockade of RRV cell binding and infection by GST–VP5* depended entirely upon the presence of VP5* D308 and G309. Thus, recombinant GST–VP5* shares with infectious RRV a DGE-dependence for cell binding. Taken in conjunction with GST–VP5* binding to the 2 I domain, which was also eliminated by the D308A and G309A mutations (Graham et al., 2003), this is clear evidence that VP5* of infectious RRV binds cellular 21 via the 2 I domain in an interaction dependent upon the presence of VP5* residues D308 and G309. Infection by RRV and Wa rotaviruses was blocked to a similar degree by RRV VP5* and depended similarly on the presence of VP5* D308 and G309. This is the first time that any competition with Wa for infectivity by a protein from RRV has been demonstrated. Wa blocks infection by the RRV nar3 mutant, but nar3 does not affect Wa infectivity (Méndez et al., 1999). Wa binds recombinant 21 on K562 cells and Wa infectivity is substantially reduced by DGE-containing peptides (Graham et al., 2003, 2004). The new findings here are a crucial component of the accumulating evidence that Wa also binds to cellular 21 integrin via the 2 I domain utilizing VP5* residues D308 and G309. It is also possible that GST–VP5* may inhibit infectious virus binding or infection through the peripheral membrane domain, the hydrophobic domain or an undefined cell-binding domain, if these functions are dependent on the presence of D308 and G309 or prior VP5* binding to 21.
Apparently conflicting findings on the ability of rotaviruses to bind 21 are reconciled by the demonstration that VP5* bound to cells at 37 °C, but not 4 °C, inhibits virus infectivity
The blockade of virus binding and infection by RRV GST–VP5* shown in Figs 3 and 4 contrasts with earlier reports that RRV binding or infection in MA104 cells is unaffected by recombinant RRV GST–VP5*. RRV GST–VP5* was reported to inhibit RRV infectivity only when MA104 cells were neuraminidase-treated (Zárate et al., 2000b). As the cell-binding partner(s) for VP5* and virus were not identified, the role of 21 was not investigated. These earlier studies were performed using MA104 cells treated with VP5* at 4 °C (Zárate et al., 2000a, b, 2003, 2004). In agreement with these reports, it was found here (Fig. 3) that treatment of MA104 cell monolayers with GST–VP5* at 4 °C had no effect on RRV infection. However, we demonstrated that treatment with GST–VP5* at 37 °C resulted in dose-dependent inhibition of RRV and Wa infection (Fig. 4). RRV binding to 21 on 2-K562 cells was also almost completely blocked by GST–VP5* at 37 °C (Fig. 3). This GST–VP5* bound 21 on 2-K562 cells at 4 °C by flow cytometry (Fig. 1) and binding of a similar construct to MA104 cells at 4 °C was partially dependent on 21 (Zárate et al., 2003); thus, the inability of GST–VP5* to block infection when added at 4 °C was not due to a lack of GST–VP5* binding to cellular 21 at this temperature.
It is clear from the above that rotavirus cell binding and infection should be measured using cells in their natural state of adhesion and at physiologically relevant temperatures when possible, and that results of experiments conducted under other conditions should be interpreted with caution. The inability of RRV VP5* at 4 °C to compete with RRV binding that was reported previously was interpreted to mean that RRV binds to the cell mainly through VP8*, as RRV VP8* at 4 °C was found to reduce RRV cell binding (Zárate et al., 2003). However, the demonstration (Fig. 3) that RRV VP5* competes at 37 °C with RRV for 21 binding is evidence that RRV binds to the cell through VP5* recognition of 21. To explain the inability of RRV VP5* at 4 °C to compete with RRV infectivity, it was proposed that RRV can attach to the cell via VP8* and then efficiently displace already bound recombinant VP5* (Zárate et al., 2000b). However, it was shown in Figs 3 and 4 that VP5* effectively competes with RRV for cell binding and infection at 37 °C, so VP5* is not displaced by RRV under these conditions. In fact, the reverse is the case. In addition, VP5* competition with RRV is for binding to 21 (Fig. 3), definitively establishing cellular 21 as a VP5* ligand that is important for rotavirus cell binding and infectivity. The conclusion that trypsin activation is necessary for RRV nar3 mutant cell attachment via VP5* also needs to be re-evaluated, as RRV GST–VP5* was reacted with cells at 4 °C in that study (Zárate et al., 2004).
Implications of the temperature dependence on the ability of VP5* to compete with rotavirus infectivity
The lack of GST–VP5* competition with rotavirus infectivity at 4 °C shows that interaction between GST–VP5* and 21, which results in blockade of virus infection, requires energy and/or active cellular processes. A likely scenario is that VP5* bound to cellular 21 undergoes conformational change between 4 and 37 °C to a structure presenting the DGE sequence analogously to virions primed to engage 21. Virions used in cell-binding studies are trypsin-activated at 37 °C, which primes virus for entry by triggering a rearrangement stabilizing the VP4 spikes as upright dimers (Crawford et al., 2001). A further, unknown event during entry has been proposed to trigger a second structural rearrangement in VP5* to produce the trimeric form seen in a crystal structure of VP5*. This second rearrangement could involve a 180° rotation of the VP5* antigen domain, including the DGE sequence (Dormitzer et al., 2004). GST–VP5* bound to 21 may take up the primed conformation, or a conformation involved in the second rearrangement, at 37 °C but not at 4 °C. Also, 21 undergoes conformational changes and lateral movement in the cell membrane at 37 °C, including activation, that may facilitate stable VP5* binding in the correct conformation for inhibition of virus–cell binding and infection (Luque et al., 1996). Supporting this, incubation of cells with anti-2 antibodies at 37 °C but not at 4 °C results in antibody blockade of RRV cell binding, and activation of 21 at 37 °C increases infectious SA11 rotavirus binding to 21 (Graham et al., 2003).
Our results show that virion VP5* binds cellular 21, an important receptor for rotavirus infection, and that VP5* additionally binds cells independently of 21. Rotavirus cell attachment and entry also involve virus recognition of several other cellular components. Further analysis of these processes is important for an understanding of rotavirus tropism and pathogenesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Ciarlet, M., Crawford, S. E., Cheng, E., Blutt, S. E., Rice, D. A., Bergelson, J. M. & Estes, M. K. (2002a). VLA-2 (21) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment. J Virol 76, 1109–1123.
Ciarlet, M., Ludert, J. E., Iturriza-Gómara, M., Liprandi, F., Gray, J. J., Desselberger, U. & Estes, M. K. (2002b). Initial interaction of rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin. J Virol 76, 4087–4095.
Clark, S. M., Roth, J. R., Clark, M. L., Barnett, B. B. & Spendlove, R. S. (1981). Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. J Virol 39, 816–822.
Coulson, B. S. & Kirkwood, C. (1991). Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus monotype. J Virol 65, 5968–5974.
Coulson, B. S., Fowler, K. J., Bishop, R. F. & Cotton, R. G. (1985). Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates. J Virol 54, 14–20.
Coulson, B. S., Tursi, J. M., McAdam, W. J. & Bishop, R. F. (1986). Derivation of neutralizing monoclonal antibodies to human rotaviruses and evidence that an immunodominant neutralization site is shared between serotypes 1 and 3. Virology 154, 302–312.[CrossRef][Medline]
Coulson, B. S., Londrigan, S. L. & Lee, D. J. (1997). Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells. Proc Natl Acad Sci U S A 94, 5389–5394.
Crawford, S. E., Labbe, M., Cohen, J., Burroughs, M. H., Zhou, Y. J. & Estes, M. K. (1994). Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol 68, 5945–5952.
Crawford, S. E., Mukherjee, S. K., Estes, M. K., Lawton, J. A., Shaw, A. L., Ramig, R. F. & Prasad, B. V. V. (2001). Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol 75, 6052–6061.
Delorme, C., Brüssow, H., Sidoti, J., Roche, N., Karlsson, K.-A., Neeser, J.-R. & Teneberg, S. (2001). Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope. J Virol 75, 2276–2287.
Denisova, E., Dowling, W., LaMonica, R., Shaw, R., Scarlata, S., Ruggeri, F. & Mackow, E. R. (1999). Rotavirus capsid protein VP5* permeabilizes membranes. J Virol 73, 3147–3153.
Dormitzer, P. R., Sun, Z. Y., Wagner, G. & Harrison, S. C. (2002). The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J 21, 885–897.[CrossRef][Medline]
Dormitzer, P. R., Nason, E. B., Prasad, B. V. V. & Harrison, S. C. (2004). Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature 430, 1053–1058.[CrossRef][Medline]
Dowling, W., Denisova, E., LaMonica, R. & Mackow, E. R. (2000). Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain. J Virol 74, 6368–6376.
Espejo, R. T., López, S. & Arias, C. (1981). Structural polypeptides of simian rotavirus SA11 and the effect of trypsin. J Virol 37, 156–160.
Estes, M. K., Graham, D. Y. & Mason, B. B. (1981). Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J Virol 39, 879–888.
Falconer, M. M., Gilbert, J. M., Roper, A. M., Greenberg, H. B. & Gavora, J. S. (1995). Rotavirus-induced fusion from without in tissue culture cells. J Virol 69, 5582–5591.[Abstract]
Fiore, L., Greenberg, H. B. & Mackow, E. R. (1991). The VP8 fragment of VP4 is the rhesus rotavirus hemagglutinin. Virology 181, 553–563.[CrossRef][Medline]
Fukudome, K., Yoshie, O. & Konno, T. (1989). Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption. Virology 172, 196–205.[CrossRef][Medline]
Gilbert, J. M. & Greenberg, H. B. (1998). Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without. J Virol 72, 5323–5327.
Golantsova, N. E., Gorbunova, E. E. & Mackow, E. R. (2004). Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability. J Virol 78, 2037–2044.
Graham, K. L., Halasz, P., Tan, Y., Hewish, M. J., Takada, Y., Mackow, E. R., Robinson, M. K. & Coulson, B. S. (2003). Integrin-using rotaviruses bind 21 integrin 2 I domain via VP4 DGE sequence and recognize X2 and V3 by using VP7 during cell entry. J Virol 77, 9969–9978.
Graham, K. L., Zeng, W., Takada, Y., Jackson, D. C. & Coulson, B. S. (2004). Effects on rotavirus cell binding and infection of monomeric and polymeric peptides containing 21 and x2 integrin ligand sequences. J Virol 78, 11786–11797.
Graham, K. L., Fleming, F. E., Halasz, P., Hewish, M. J., Nagesha, H. S., Holmes, I. H., Takada, Y. & Coulson, B. S. (2005). Rotaviruses interact with 47 and 41 integrins by binding the same integrin domains as natural ligands. J Gen Virol 86, 3397–3408.
Guerrero, C. A., Méndez, E., Zárate, S., Isa, P., López, S. & Arias, C. F. (2000). Integrin v3 mediates rotavirus cell entry. Proc Natl Acad Sci U S A 97, 14644–14649.
Guo, C. T., Nakagomi, O., Mochizuki, M. & 7 other authors (1999). Ganglioside GM(1a) on the cell surface is involved in the infection by human rotavirus KUN and MO strains. J Biochem 126, 683–688.
Halasz, P., Fleming, F. E. & Coulson, B. S. (2005). Evaluation of specificity and effects of monoclonal antibodies submitted to the Eighth Human Leucocyte Differentiation Antigen Workshop on rotavirus-cell attachment and entry. Cell Immunol 236, 179–187.[CrossRef][Medline]
Hewish, M. J., Takada, Y. & Coulson, B. S. (2000). Integrins 21 and 41 can mediate SA11 rotavirus attachment and entry into cells. J Virol 74, 228–236.
Jolly, C. L., Beisner, B. M. & Holmes, I. H. (2000). Rotavirus infection of MA104 cells is inhibited by Ricinus lectin and separately expressed single binding domains. Virology 275, 89–97.[CrossRef][Medline]
Jolly, C. L., Beisner, B. M., Ozser, E. & Holmes, I. H. (2001a). Non-lytic extraction and characterisation of receptors for multiple strains of rotavirus. Arch Virol 146, 1307–1323.[CrossRef][Medline]
Jolly, C. L., Huang, J.-A. & Holmes, I. H. (2001b). Selection of rotavirus VP4 cell receptor binding domains for MA104 cells using a phage display library. J Virol Methods 98, 41–51.[CrossRef][Medline]
Kaljot, K. T., Shaw, R. D., Rubin, D. H. & Greenberg, H. B. (1988). Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis. J Virol 62, 1136–1144.
Kirkwood, C. D., Bishop, R. F. & Coulson, B. S. (1998). Attachment and growth of human rotaviruses RV-3 and S12/85 in Caco-2 cells depend on VP4. J Virol 72, 9348–9352.
Liakatos, A., Kiefel, M. J., Fleming, F., Coulson, B. & von Itzstein, M. (2006). The synthesis and biological evaluation of lactose-based sialylmimetics as inhibitors of rotaviral infection. Bioorg Med Chem 14, 739–757.[CrossRef][Medline]
Londrigan, S. L., Hewish, M. J., Thomson, M. J., Sanders, G. M., Mustafa, H. & Coulson, B. S. (2000). Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins. J Gen Virol 81, 2203–2213.
Londrigan, S. L., Graham, K. L., Takada, Y., Halasz, P. & Coulson, B. S. (2003). Monkey rotavirus binding to 21 integrin requires the 2 I domain and is facilitated by the homologous 1 subunit. J Virol 77, 9486–9501.
Ludert, J. E., Feng, N., Yu, J. H., Broome, R. L., Hoshino, Y. & Greenberg, H. B. (1996). Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo. J Virol 70, 487–493.[Abstract]
Ludert, J. E., Ruiz, M. C., Hidalgo, C. & Liprandi, F. (2002). Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation. J Virol 76, 6643–6651.
Luque, A., Gómez, M., Puzon, W., Takada, Y., Sánchez-Madrid, F. & Cabañas, C. (1996). Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common 1 chain. J Biol Chem 271, 11067–11075.
Méndez, E., Arias, C. F. & López, S. (1993). Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture. J Virol 67, 5253–5259.
Méndez, E., López, S., Cuadras, M. A., Romero, P. & Arias, C. F. (1999). Entry of rotaviruses is a multistep process. Virology 263, 450–459.[CrossRef][Medline]
Nagesha, H. S. & Holmes, I. H. (1991). VP4 relationships between porcine and other rotavirus serotypes. Arch Virol 116, 107–118.[CrossRef][Medline]
Pesavento, J. B., Crawford, S. E., Roberts, E., Estes, M. K. & Prasad, B. V. V. (2005). pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization. J Virol 79, 8572–8580.
Rolsma, M. D., Kuhlenschmidt, T. B., Gelberg, H. B. & Kuhlenschmidt, M. S. (1998). Structure and function of a ganglioside receptor for porcine rotavirus. J Virol 72, 9079–9091.
Warner, S., Hartley, C. A., Stevenson, R. A., Ficorilli, N., Varrasso, A., Studdert, M. J. & Crabb, B. S. (2001). Evidence that Equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding. J Virol 75, 9274–9281.
Yeager, M., Dryden, K. A., Olson, N. H., Greenberg, H. B. & Baker, T. S. (1990). Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J Cell Biol 110, 2133–2144.
Zárate, S., Espinosa, R., Romero, P., Guerrero, C. A., Arias, C. F. & López, S. (2000a). Integrin 21 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3. Virology 278, 50–54.[CrossRef][Medline]
Zárate, S., Espinosa, R., Romero, P., Méndez, E., Arias, C. F. & López, S. (2000b). The VP5 domain of VP4 can mediate attachment of rotaviruses to cells. J Virol 74, 593–599.
Zárate, S., Cuadras, M. A., Espinosa, R., Romero, P., Juárez, K. O., Camacho-Nuez, M., Arias, C. F. & López, S. (2003). Interaction of rotaviruses with Hsc70 during cell entry is mediated by VP5. J Virol 77, 7254–7260.
Zárate, S., Romero, P., Espinosa, R., Arias, C. F. & López, S. (2004). VP7 mediates the interaction of rotaviruses with integrin v3 through a novel integrin-binding site. J Virol 78, 10839–10847.
Received 3 October 2005;
accepted 18 January 2006.
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