1 Integrin Mediates Internalization of Mammalian R
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病菌学杂志 2006年第6期
Departments of Microbiology and Immunology Pathology Pediatrics
Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
The Scripps Research Institute, La Jolla, California 92037
Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
ABSTRACT
Reovirus infection is initiated by interactions between the attachment protein 1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein 2, which serves as the structural base for 1, suggests that integrins mediate reovirus endocytosis. A 1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a 1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in 1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing 1. However, reovirus bound equivalently to cells that differed in levels of 1 expression, suggesting that 1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into 1-deficient cells was substantially diminished in comparison to viral uptake into 1-expressing cells. These data provide evidence that 1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.
INTRODUCTION
Viral attachment and cell entry are key determinants of target cell selection in the infected host and thus play important roles in pathogenesis. Many viruses interact with multiple cell surface molecules to mediate the processes of attachment and internalization (68). For example, human immunodeficiency virus uses CD4 to bind the cell surface and chemokine receptors to facilitate the conformational alterations in envelope glycoproteins that culminate in fusion of the viral envelope and cell membrane (35). Receptors that serve as initial binding sites have been identified for many viruses (25). However, little is known about the postattachment events that lead to nonenveloped virus internalization, in particular those that mediate virus uptake into the endocytic pathway.
Mammalian reoviruses are large, nonenveloped, double-stranded RNA-containing viruses that infect a variety of mammalian species. Following attachment to target cells, reoviruses are internalized by receptor-mediated endocytosis (3, 13, 14, 72), which is mostly likely to be clathrin dependent (31). Proteolytic disassembly in endosomes leads to removal of outer capsid protein 3 and cleavage of outer capsid protein μ1 (3, 14, 27, 72). The resultant disassembly intermediate formed by these events, the infectious subvirion particle (ISVP), is capable of penetrating endosomal membranes in a μ1-dependent manner to release the transcriptionally active viral core particle into the cytoplasm (17, 18, 58), where viral replication takes place. Cellular determinants of reovirus receptor-mediated internalization following attachment and preceding uncoating are poorly defined.
We previously identified junctional adhesion molecule A (JAM-A) as a serotype-independent receptor for reovirus (5, 16, 34). JAM-A is a type 1 transmembrane protein expressed in a variety of cell types, including polarized endothelial and epithelial cells and circulating leukocytes (52, 55, 81). JAM-A interacts with several scaffolding proteins and cytoplasmic adaptor molecules (6, 28, 29) and is hypothesized to play an important role in maintaining the barrier function of epithelial junctions (48, 52, 55, 60). JAM-A is phosphorylated during platelet activation and required for mitogen-activated protein kinase activation following treatment of endothelial cells with basic fibroblast growth factor (57). These data indicate that JAM-A is intimately associated with cytoskeletal and signaling machinery, which raises the possibility that reovirus binding to JAM-A mediates cytoskeletal rearrangement or signaling events to facilitate virus internalization.
The attachment mechanisms of reovirus and adenovirus are remarkably similar (70, 71). The trimeric attachment proteins of both viruses, 1 and fiber, respectively, are structural homologues and fold using a highly unusual triple -spiral motif (10, 20, 83). The globular head domains of these molecules are formed from eight-stranded -barrels with identical interstrand connectivity (70). The receptors for 1 and fiber, JAM-A (5) and coxsackievirus and adenovirus receptor (CAR) (7), respectively, are two-domain, immunoglobulin superfamily proteins that form homodimers using analogous molecular surfaces (71). Also, both JAM-A and CAR localize to tight junctions in polarized epithelial cells (23, 52, 55, 60). Remarkably, reovirus and adenovirus engage their respective receptors by thermodynamically favored disruption of receptor homodimers (34, 53).
Despite mediating high-affinity attachment of adenovirus to cells, engagement of CAR does not permit efficient adenovirus internalization. Instead, adenovirus entry is enhanced by high-avidity interactions of the viral penton base complex with integrins, including v3 and v5 (80). Integrins are heterodimeric cell surface molecules that consist of and subunits (43). Integrins function to mediate cellular adhesion to the extracellular matrix, regulate cellular trafficking, and transduce both outside-in and inside-out signaling events (42). In addition to adenovirus, several other pathogenic microorganisms have usurped the adhesion and signaling properties of integrins to bind or enter host cells (1, 8, 32, 39-41, 44, 49).
To define the molecular basis of reovirus internalization, we first tested the capacity of a JAM-A mutant lacking a cytoplasmic tail to support reovirus attachment and infection. We found that while JAM-A is necessary for efficient attachment to cells, the JAM-A cytoplasmic tail is not required for reovirus infection. Given the mechanistic conservation of reovirus and adenovirus attachment strategies and the observation that reovirus outer capsid protein 2 contains the conserved integrin-binding sequences Arg-Gly-Asp (RGD) and Lys-Gly-Glu (KGE), we tested the role of integrins in reovirus internalization. We found that infection by reovirus virions is inhibited by antibodies specific for 1 integrin. In addition, cells deficient in 1 integrin have a diminished susceptibility to reovirus infection due to a postattachment block to viral entry. Together, these data indicate that, following attachment to JAM-A, 1 integrin facilitates internalization of reovirus into cells. Our findings further demonstrate that two seemingly unrelated viruses utilize distinct cellular molecules to mediate attachment and internalization in a remarkably similar manner.
MATERIALS AND METHODS
Cells, viruses, and antibodies. Spinner-adapted murine L929 (L) cells were grown in either suspension or monolayer cultures in Joklik's modified Eagle's minimal essential medium (Irvine Scientific, Santa Ana, CA) supplemented to contain 5% fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin per ml, 100 U of streptomycin per ml, and 0.25 mg amphotericin per ml (Gibco Invitrogen Corp., Grand Island, NY). Chinese hamster ovary (CHO) cells were maintained in Ham's F12 medium (Irvine Scientific) supplemented to contain 10% fetal bovine serum, 100 U of penicillin per ml, and 100 U of streptomycin per ml. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Gibco Invitrogen Corp.) and supplemented as described for CHO cells. Primary cultures of chicken embryo fibroblasts (CEFs) were obtained from Paul Spearman (Vanderbilt University) and maintained in medium 199 with Earle's salts and 2.2 mg sodium bicarbonate per ml (Gibco Invitrogen Corp.) supplemented to contain 5% fetal bovine serum, 10% tryptose phosphate broth, 1% chicken serum (Gibco Invitrogen Corp.), and antibiotics as described for CHO cells. GD25 and GD251A cells were obtained from Deane Mosher (University of Wisconsin, Madison) (78) and maintained as described for HeLa cells. Medium for GD251A cells was supplemented to contain 10 μg of puromycin (Sigma-Aldrich, St. Louis, MO) per ml to maintain 1 integrin expression.
Reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) are laboratory stocks. Working stocks of virus were prepared by plaque purification and passage in L cells (75). Purified virions were generated from second-passage L-cell lysate virus stocks. Virus was purified from infected cell lysates by Freon extraction and CsCl gradient centrifugation as described (36). Bands corresponding to the density of reovirus particles (1.36 g/cm3) were collected and dialyzed against virion storage buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl [pH 7.4]). Reovirus particle concentration was determined by the equivalence of 1 unit of optical density at 260 nm to 2.1 x 1012 particles (69).
Viral infectivity titers were determined by either plaque assay (75) or fluorescent focus assay (4). ISVPs were generated by treatment of 2 x 1011 virion particles per ml with 2 mg of -chymotrypsin (Sigma-Aldrich) per ml in a volume of 100 μl virion storage buffer at 37°C for 30 min (2). Reactions were terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 1.0 mM. Purified T1L virions in carbonate-bicarbonate buffer (Sigma-Aldrich) were fluoresceinated by incubation with 50 μg fluorescein isothiocyanate (FITC) (Pierce, Rockford, IL) per ml at room temperature for 1 h (38). Excess FITC was removed by exhaustive dialysis against phosphate-buffered saline (PBS).
Immunoglobulin G (IgG) fractions of rabbit antisera raised against T1L and T3D (79) were purified by protein A-Sepharose as previously described (4). Fluorescently conjugated secondary Alexa antibodies were obtained from Molecular Probes (Invitrogen, San Diego, CA). The human JAM-A (hJAM-A)-specific monoclonal antibody (MAb) J10.4 and control mouse ascites were provided by Charles Parkos (Emory University School of Medicine) (52), and the murine JAM-A (mJAM-A)-specific MAb H202-106-7-4 was provided by Beat Imof (University of Geneva). The human 2-specific MAb AA10 (IgM) (8) and human 1-specific MAb DE9 (IgG1) (8) were used as diluted ascites. Human integrin-specific MAbs MAB1980 (v), MAB1973 (1), MAB2057 (3), MAB1378 (6), MAB1976 (v3), and MAB1961Z (v5) were purchased from Chemicon International (Temecula, CA). Antibody BIIG2 (5) (Developmental Hybridoma Studies Bank, University of Iowa, Iowa City, IA) was provided by John Williams (Vanderbilt University). Function-blocking human 2-specific MAb 6F1 was provided by Richard Bankert (State University of New York at Buffalo). Function-blocking murine 1 MAb CD29 (IgM) and hamster IgM isotype control were purchased from BD Biosciences Pharmingen (San Jose, CA). Murine 1-specific MAb MAB1997 (Chemicon) and human 1-specific MAb MAB2253Z (Chemicon) were used to assess expression of 1 integrin on GD25 and GD251A cells and HeLa cells, respectively, by flow cytometry. ICAM-1-specific MAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies used for flow cytometric analysis of HeLa cells are shown in Table 1.
Sequence analysis. The sequences of the reovirus 2-encoding L2 gene from strains T1L (NC_004259), type 2 Jones (T2J) (NC_004260), T3D (NC_004275), T1Neth85 (AF378004), T2SV59 (AF378006), T3C9 (AF378007), T3C18 (AF378008), T3C87 (AF378009), and T3C93 (AF378010) were aligned using the protein sequence alignment algorithm in MacVector, version 8.0.1 (Accelrys, San Diego, CA).
Plasmid constructs. Human JAM-A was subcloned into expression plasmid pcDNA3.1+ (Invitrogen) (34). Truncation mutant JAM-A-CT was generated by PCR using full-length JAM-A cDNA as the template. Amino acids 1 to 260 (261-299) were cloned and appended with a stop codon using T7 primer and 5'-TACGGGATCCTCAGGCAAACCAGATGCC-3' as the forward and reverse primers, respectively. The gene-specific primer encompasses nucleotides 981 to 995 of the JAM-A cDNA. The PCR product was digested with BamHI (recognition site underlined in the reverse primer sequence) and subcloned into the complementary restriction sites of pcDNA3.1+. Fidelity of cloning was confirmed by automated sequencing. Plasmid constructs encoding murine integrin v (33) and 2 (30) were previously described. A cDNA encoding murine 1 integrin cloned into the EcoRI site of pGEM1 was obtained from Richard Hynes (Massachusetts Institute of Technology) (D. W. DeSimone, V. Patel, and R. O. Hynes, unpublished). Integrin cDNAs were subcloned into the expression plasmid pcDNA3.1+.
Transient transfection of CHOs and CEFs. Monolayers of cells in a 24-well plate (Costar, Cambridge, MA) were transfected with empty vector or plasmids encoding receptor constructs by using Lipofectamine Plus reagent (Invitrogen). Cells were incubated for 24 h to allow receptor expression prior to adsorption with either reovirus virions or ISVPs for infectivity studies.
Flow cytometric analysis. Surface expression of integrin subunits on HeLa cells was determined by flow cytometry. Cells were detached from plates by using PBS-EDTA (20 mM EDTA). Cells were washed and centrifuged at 2,000 x g to form a pellet, resuspended with integrin-specific or control antibodies in PBS- bovine serum albumin (BSA) (Sigma-Aldrich) (5% BSA), and incubated at 4°C for 1 h. Cells were washed twice and incubated with an appropriate secondary antibody conjugated to R-phycoerythrin (BD Biosciences Pharmingen) at 4°C for 1 h. Cells were washed, resuspended in PBS, and analyzed by flow cytometry. Results were analyzed using the Windows Multiple Document 2.8 flow cytometry application (The Scripps Research Institute, La Jolla, CA).
The mean fluorescence intensity was measured for an average of 14,000 gated events for cells treated with control or integrin-specific antibodies. Events were gated relative to cells stained with an appropriate secondary antibody conjugated to phycoerythrin. Reovirus binding to GD25 and GD251A cells was analyzed by adsorbing cells with 2 x 1011 FITC-labeled particles of strain T1L at 4°C for 1 h. Cells were washed and analyzed by flow cytometry.
Fluorescent focus assay of viral infection. Cells were plated in 24-well or 96-well plates (Costar) and adsorbed with virus at various multiplicities of infection (MOIs) at either 4°C or room temperature for 30 to 60 min. Inocula were removed, cells were washed, and complete medium was added. Infected cells were incubated at 37°C for 16 to 24 h to allow a single cycle of viral replication. Cells were fixed with methanol at –20°C for at least 30 min. Fixed cells were incubated with PBS-BSA (5% BSA) for at least 15 min, followed by incubation with reovirus-specific polyclonal antiserum (1:500) in PBS-Triton X-100 (0.5% Triton X-100) at room temperature for 1 h. Cells were washed twice and incubated with an Alexa 488- or 546-labeled anti-rabbit IgG (1:1,000) in PBS-Triton X-100 (0.5% Triton X-100) at room temperature for 1 h.
Cells were washed twice and visualized by indirect immunofluorescence at a magnification of 20x using an Axiovert 200 fluorescence microscope (Carl Zeiss, New York, NY). Infected cells (fluorescent focus units [FFU]) were identified by diffuse cytoplasmic fluorescence staining that was excluded from the nucleus. Reovirus-infected cells were quantified by counting random fields of view of equivalently confluent monolayers for three to five fields of view for triplicate wells or by counting the entire well for triplicate wells (4).
Confocal imaging of reovirus internalization. GD25 and GD251A cells were plated on coverslips in 24-well plates. Cells were chilled at 4°C for 45 min prior to infection, washed with PBS, adsorbed with 8 x 105 particles per cell of T1L virions in gelatin saline, and returned to 4°C for 1 h. The MOI used was the minimum number of particles required to detect signal by confocal immunofluorescence microscopy at early time points postinfection. Cells were either washed and fixed or nonadherent reovirus was aspirated and replaced with warm Dulbecco's modified Eagle's medium and returned to 37°C. At 10-min intervals, cells were washed with PBS and fixed with 4% formaldehyde for 20 min. Excess formaldehyde was quenched with an equal amount of 0.1 M glycine, followed by washing with PBS. Cells were treated with 1% Triton X-100 for 5 min and incubated with PBS-BGT (PBS, 0.5% BSA, 0.1% glycine, and 0.05% Tween 20) for 10 min. Cells were incubated with reovirus-specific polyclonal antiserum (1:500) in PBS-BGT for 1 h and washed with PBS-BGT. Cells were stained with donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 488 (Molecular Probes) (1:500) to visualize reovirus, phalloidin conjugated to Alexa Fluor 546 (Molecular Probes) (1:100) to visualize actin, and TO-PRO 3 conjugated to Alexa Fluor 642 (Molecular Probes) (1:1,000) to visualize DNA. Cells were incubated for 1 h with secondary antibodies and fluorescent probes in PBS-BGT and washed with PBS-BGT. Coverslips were removed from wells and placed on slides using Prolong Anti-Fade mounting medium (Molecular Probes). Images were captured on a Zeiss LSM 510 laser-scanning confocal microscope using LSM 510 software.
Virus internalization was quantified by enumerating fluorescent particles localized at the cell periphery and particles internalized into the cytoplasm to determine the total number of fluorescent particles per cell. Ten cells were analyzed for each time point. The number of internalized particles was measured as a percentage of the total number of particles per cell.
Statistical analysis. Means of triplicate samples were compared by using an unpaired Students' t test (Microsoft Excel, Redmond, WA). P values of <0.05 were considered statistically significant.
RESULTS
The JAM-A cytoplasmic tail is dispensable for reovirus infection. JAM-A is a serotype-independent reovirus receptor with a cytoplasmic tail known to interact with a variety of proteins (6, 28, 29). To determine whether the JAM-A cytoplasmic tail is required for reovirus entry, we generated a JAM-A cytoplasmic tail deletion mutant (JAM-A-CT) and tested its capacity to support reovirus infection following transfection of CHO cells. CHO cells do not express detectable levels of JAM-A (55, 60) and are poorly permissive for reovirus infection (34). Cells were transiently transfected with plasmids encoding full-length JAM-A, JAM-A-CT, or empty vector as a control. Equivalent cell surface expression of transfected constructs was confirmed by flow cytometry (data not shown).
The capacity of reovirus to infect CHO cells following transfection with the JAM-A constructs was tested using reovirus fluorescent focus assays. Following transient transfection of CHO cells with empty vector, JAM-A, or JAM-A-CT, cells were adsorbed with reovirus strains T1L and T3D and scored for infection by indirect immunofluorescence at 20 h postinfection (Fig. 1). Expression of either full-length or truncated JAM-A was sufficient to allow reovirus infection of CHO cells, permitting viral protein production of both type 1 and type 3 reovirus strains. These results indicate that the JAM-A cytoplasmic tail is not required for efficient reovirus attachment and infection.
Reovirus outer capsid proteins contain integrin-binding sequences. Structural and functional analyses indicate that reovirus and adenovirus share remarkably similar mechanisms of attachment (70, 71). To determine whether reovirus outer capsid proteins contain sequences that could potentially engage integrins, we performed a search for integrin-binding motifs in the 1, 3, μ1, and 2 proteins, which form the reovirus outer capsid (26). We identified two common integrin-binding motifs, RGD and KGE, in the deduced amino acid sequence of the 2 protein (Fig. 2). The RGD motif is conserved in all reovirus strains for which sequence information is available (15, 67); the KGE motif is conserved in all of those strains except T2J (15, 67). The 2 protein is a component of the reovirus outer capsid and core (26). It is structurally arranged as a pentamer at the virion fivefold axes of symmetry and forms the base for attachment protein 1 (26, 63). The presence of conserved integrin-binding motifs in the reovirus 2 protein led us to test whether reovirus utilizes integrins to mediate internalization.
An antibody specific for 1 integrin inhibits reovirus infection of HeLa cells. To determine whether integrins are required for reovirus infection, we first used flow cytometry to analyze integrin expression on the surface of HeLa cells. HeLa cells were incubated with integrin-specific MAbs and a phycoerythrin-labeled secondary antibody (Table 1). RGD-binding integrin subunits 3, 5, v, and 1 and KGE-binding integrin subunits 1, 2, 6, and 1 (43) were detected on HeLa cells at levels above those in control antibody-treated cells. RGD-binding integrin heterodimer v5 also was detected at levels above that of the control, while there was low-level expression of v3. Thus, HeLa cells express both RGD- and KGE-binding integrins.
To assess a role for integrins in reovirus replication, we tested antibodies specific for the RGD- and KGE-binding integrins expressed on HeLa cells for the capacity to block reovirus infection. HeLa cells were incubated with integrin-specific and control antibodies prior to adsorption with reovirus virions. Viral infection was detected by indirect immunofluorescence (Fig. 3A). We found that 1-specific MAb DE9 resulted in a 50% reduction in infection (P < 0.05), while antibodies specific for the other integrin subunits expressed on HeLa cells had no effect. Control antibodies produced anticipated effects; JAM-A-specific MAb J10.4 inhibited infection, whereas ICAM-specific MAb (data not shown) or control mouse ascites (Fig. 3A) did not. The effect of MAb DE9 was dose dependent (Fig. 3B), providing further evidence that the inhibition of infection was dependent on integrin blockade.
To determine whether particular subunits pair with 1 integrin to facilitate reovirus infection, we tested whether treatment with integrin-specific antibodies was capable of enhancing the inhibitory effect of 1 integrin-specific MAb DE9 on reovirus infection. We also tested whether antibodies specific for other integrin subunits expressed on HeLa cells, 3 and 5, were capable of infection blockade. HeLa cells were treated with MAb DE9 in combination with other integrin-specific antibodies prior to adsorption with reovirus virions (Fig. 3C). While treatment of HeLa cells with MAb DE9 resulted in a 50% reduction in reovirus infection, none of the other integrin-specific antibodies tested reduced reovirus infection to a greater extent than that resulting from treatment with DE9 alone. These results suggest that the integrin epitope bound by reovirus is blocked by 1-specific MAb DE9 and not by the other MAbs used in these experiments.
JAM-A MAb J10.4 blocks reovirus infection 90% (Fig. 3A). To determine whether the residual level of infection in the presence of MAb J10.4 was dependent on reovirus interactions with 1 integrin, we treated HeLa cells with JAM-A-specific MAb J10.4 in combination with MAb DE9 prior to adsorption with reovirus virions (Fig. 3D). Treatment of HeLa cells with MAb J10.4 and MAb DE9 completely abrogated reovirus infection, indicating that the effect of JAM-A blockade is enhanced when 1 integrin is not available for interactions with reovirus. Treatment with MAb DE9 did not significantly inhibit infection by ISVPs (Fig. 3D), suggesting that viral attachment is not affected by 1 integrin blockade. Taken together, these results support the conclusion that a 1-specific antibody blocks reovirus infection at a step subsequent to attachment but prior to uncoating, implicating 1 integrin in reovirus internalization.
Transient transfection of integrin cDNAs allows reovirus infection of JAM-A-expressing CEFs. Ectopic expression of JAM-A in CEFs rescues infection by reovirus ISVPs but not by virions (5), suggesting that these cells exhibit a cell-specific block at the entry or uncoating phases of reovirus infection. To test the capacity of integrins to confer infection of CEFs by reovirus virions, CEFs were transiently transfected with a JAM-A-encoding plasmid in the presence or absence of murine v, 2, or 1 integrin-encoding plasmids singly or in pairs. Transfected cells were infected with reovirus virions or ISVPs, and infection was assessed by indirect immunofluorescence (Fig. 4). Expression of 1 integrin paired with either of the murine integrin subunits provided an approximately fourfold enhancement of infection by reovirus virions in comparison to that in cells transfected with JAM-A alone. These data suggest that 1 integrin expression complements a reovirus cell entry defect in CEFs and provide further support for the involvement of 1 integrin in reovirus internalization.
Cells deficient in 1 integrin have a decreased capacity to support reovirus infection. To further assess a role for 1 integrin in reovirus infection, we tested the capacity of reovirus to infect cells deficient in the 1-integrin subunit. GD25 cells are murine embryonic stem cells derived from 1-null embryos (78). GD251A cells are GD25 cells that have been engineered to stably express 1 integrin and thus serve as an isogenic control for GD25 cells. Flow cytometric analysis confirmed that while both cells express JAM-A, only GD251A cells express 1 integrin (Fig. 5A). GD25 cells (1–/–) and GD251A cells (1+/+) (78) were adsorbed with reovirus virions or ISVPs, and infection was scored by indirect immunofluorescence (Fig. 5B). In comparison to 1+/+ cells, 1–/– cells were substantially less susceptible to infection by virions, while infection by ISVPs was equivalent in both cell types. Importantly, preincubation of 1+/+ cells with murine 1 integrin-specific MAb CD29 reduced infection in 1+/+ cells (Fig. 5C), indicating that enhancement of infection is due to expression of 1 integrin. Therefore, 1 integrin is required for efficient reovirus infection.
Reovirus binding to 1–/– and 1+/+ cells is equivalent. Equivalent infection of 1–/– and 1+/+ cells by ISVPs (Fig. 5B) suggests that reovirus is capable of efficiently binding to both cell types. To directly test this hypothesis, 1–/– and 1+/+ cells were mock treated or incubated with FITC-labeled virions and binding was assessed by flow cytometry (Fig. 6). In these experiments, we found that reovirus binds equivalently to 1–/– and 1+/+ cells. These data demonstrate a function for 1 integrin in reovirus infection at a step subsequent to viral attachment.
1 integrin enhances the efficiency of reovirus internalization. To directly assess the role of 1 integrin in reovirus internalization, 1–/– and 1+/+ cells were infected at 4°C and then warmed to 37°C over a time course concurrent with reovirus entry (2, 72). At 10-min intervals, cells were fixed, stained for indirect immunofluorescence, and examined by confocal microscopy. Representative confocal micrographic images of reovirus-infected 1–/– and 1+/+ cells are shown in Fig. 7. Immediately after viral adsorption, both cell types exhibited reovirus staining at the cell periphery. At 10 min postadsorption, some reovirus staining was observed at the cell periphery, yet intracellular staining in 1+/+ cells was also observed. At 20 and 30 min postadsorption, the majority of virions had entered the 1+/+ cells and had a perinuclear location. In sharp contrast to the findings made using 1+/+ cells, viral entry was markedly delayed in 1–/– cells, with the majority of reovirus virions remaining at the cell periphery throughout the time course. At later time points (30 min postadsorption), some virions were present within the cytoplasm, but these were the minority. These findings suggest that expression of 1 integrin enhances reovirus entry.
To quantify reovirus internalization into 1–/– and 1+/+ cells, we determined the number of internalized fluorescent particles as a percentage of the total number of fluorescent particles per cell at various times postadsorption (Fig. 8). At 0 and 10 min postadsorption, the percentage of particles internalized into 1–/– and 1+/+ cells was equivalent, 10 and 30%, respectively. However, at 20 and 30 min postadsorption, the percentage of reovirus particles internalized into 1+/+ cells was 50%, while the percentage of particles internalized into 1–/– cells was only 30% (P < 0.05) (Fig. 8). These data indicate that 1 integrin enhances reovirus entry at early times postadsorption, suggesting a direct role for 1 integrin as a reovirus internalization receptor.
DISCUSSION
In this study, we performed experiments to define the molecular determinants of reovirus internalization. We show that antibodies specific for 1 integrin inhibit reovirus infection at a postattachment step. We provide evidence that expression of 1 integrin promotes infection by reovirus virions in cells with a block to viral internalization and that viral entry is substantially diminished in cells deficient in 1 integrin expression. Together, these data provide strong evidence that 1 integrin serves as a coreceptor to mediate reovirus internalization. These findings suggest a new model for attachment and cell entry of reovirus (Fig. 9). In this model, we propose that reovirus initially interacts with cells via low-affinity binding to carbohydrate. These interactions are followed by high-affinity engagement of JAM-A, which positions the virus on the cell surface for subsequent interactions with 1 integrin to trigger viral endocytosis.
Integrins have been identified as attachment and entry receptors for several viruses, including echovirus (21) (8), foot-and-mouth disease virus (v1, v3, and v6) (9, 44, 45), hantaviruses NY-1 and Sin Nombre virus (3 integrins) (37), Kaposi sarcoma herpesvirus (31) (1), and cytomegalovirus (21, 61, and v3) (32). The Reoviridae family member rotavirus also engages a variety of integrins for attachment and cell entry. Rotavirus strains RRV, SA11, and Wa bind to the I (inserted) domain of 21 integrin via an Asp-Gly-Glu integrin-binding motif in the VP4 spike protein to effect viral attachment (39). The interactions of rotavirus outer capsid protein VP7 with integrins x2 and v3 can mediate viral entry (39, 82). Integrin 41 also can serve as a receptor for rotavirus strain SA11, which contains 41 integrin-binding sequences Leu-Asp-Val in VP7 and Ile-Asp-Ala in VP4 (41). Interestingly, like reovirus, adenovirus engages a specific cell surface protein, CAR, prior to interactions with integrins, which function subsequent to viral attachment to mediate viral endocytosis (50, 80). Therefore, the identification of 1 integrin as a reovirus internalization receptor suggests that the conservation of attachment strategies used by reovirus and adenovirus (70, 71) extends to mechanisms of internalization.
Although the specific reovirus protein required for integrin binding is not apparent from our studies, the 2 protein is a promising candidate. The 2 protein forms a pentameric turret at the virion fivefold symmetry axes and serves as the insertion site for trimers of attachment protein 1 (26). Thus, 2 is the reovirus analogue of the adenovirus penton base protein, which mediates the engagement of integrins by adenovirus (22, 80). Interestingly, 2 also contains conserved RGD and KGE motifs (15), the preferred interaction motifs for several 1 integrin heterodimers (43).
Structural information for 2 is available in the context of the reovirus core but not for the intact virion. In the core, the KGE motif is exposed on the top of the 2 turret, where it would be accessible to a receptor. The RGD motif is also surface exposed, but it appears to be less accessible. However, the 2 structure in the core may not be identical to that in the virion, as the protein undergoes major conformational changes during virion-to-core disassembly (26). Therefore, it is possible that both the RGD and KGE motifs are accessible to interactions with 1 integrin during engagement of the cell surface by the virus.
A human 1 integrin-specific antibody (DE9) reduced reovirus infection of HeLa cells by 50% (Fig. 3). Similarly, a murine 1 integrin-specific antibody (CD29) blocked infection of 1-expressing mouse embryonic stem cells by 50% (Fig. 4). Interestingly, MAb DE9 also blocks infection of echovirus (8) and cytomegalovirus (32), suggesting that an epitope in 1 integrin recognized by MAb DE9 may be a preferred binding site for multiple viruses. It is possible that the residual level of reovirus infection following 1 integrin antibody treatment is attributable to other internalization receptors on the cell surface that may be integrin or nonintegrin molecules. However, it is noteworthy that treatment of HeLa cells with both MAb DE9 and JAM-A-specific MAb J10.4 completely abolishes reovirus growth (Fig. 3D). This finding suggests that the residual infection in J10.4-treated HeLa cells is due to reovirus interactions with 1 integrin. Thus, it appears that blockade of reovirus infection by integrin-specific antibodies is inefficient because complete inhibition of virus-integrin interactions is not possible if the virus is tightly adhered to the cell surface by JAM-A.
Since antibodies specific for 3 and 5 integrins did not inhibit reovirus infection, it is likely that only 1 integrin can serve a reovirus internalization function. Antibodies specific for the integrin subunits expressed on HeLa cells did not further reduce reovirus infection following treatment with a 1 integrin-specific antibody (Fig. 3C). We envision three possible explanations for this result. First, reovirus may directly engage a ligand-binding domain of 1 integrin. Second, reovirus may utilize 1 integrin when paired with numerous subunits that have redundant functions. However, treatment of HeLa cells with a 1 integrin-specific antibody and a mixture of antibodies specific for 1, 2, 3, 5, 6, and v integrins did not diminish reovirus infection in comparison to cells treated with a 1 integrin-specific antibody alone (data not shown). Third, reovirus may engage an epitope of an integrin subunit that is not recognized by the antibodies used in our experiments. Further studies are required to define the biophysical basis of reovirus-integrin interactions.
JAM-A is required for high-affinity reovirus attachment to numerous cell types (5, 16, 34, 56). However, the JAM-A cytoplasmic tail is not necessary for viral endocytosis (Fig. 1). JAM-A likely tethers the virus to the cell surface to facilitate secondary interactions with 1 integrin (Fig. 9). This model is analogous to the mechanism of lymphocyte homing, in which adhesion molecules such as JAM-A provide initial cellular contacts to facilitate subsequent interactions with integrins for diapedesis or signaling (65). An interesting possibility is that JAM-A may be associated with 1 integrin on the host cell plasma membrane. If such were the case, initial JAM-A engagement might facilitate integrin binding, clustering, and viral endocytosis. In support of this hypothesis, JAM-A has been shown to regulate 1 integrin expression and localization (54).
The cytoplasmic domains of integrin subunits are involved in a number of signaling pathways (42). The 1 integrin cytoplasmic domain is linked to cytoskeletal proteins, including talin (62) and -actinin (59), and signaling molecules, including paxillin and focal adhesion kinase (66). In addition, the 1 integrin cytoplasmic domain contains two Asn-Pro-any residue-Tyr (NPXY) motifs (64), which are common sequence motifs in the cytoplasmic domains of many receptors and serve as recognition sites for the cellular endocytic machinery (21, 24). NPXY motifs interact with the μ2 subunit of the adaptor protein 2 complex (12, 61), which can recruit clathrin and trigger clathrin-mediated endocytosis (47).
Since clathrin-dependent mechanisms have been implicated in reovirus cell entry (31), it seems plausible that reovirus engagement of 1 integrin leads to clathrin-mediated endocytosis through signaling regulated by the 1 integrin cytoplasmic domain. It is noteworthy that Kaposi's sarcoma-related herpesvirus binding to 31 integrin (1) and cytomegalovirus binding to 1 integrin (32) activate focal adhesion kinase. In addition, adenovirus engagement of v integrins induces activation of phosphoinositide-3-OH kinase, which is required for adenovirus endocytosis (51).
Identification of 1 integrin as a receptor that triggers reovirus entry raises the possibility that coreceptor binding influences reovirus tropism and disease. Reovirus serotypes differ in mechanisms of spread, tropism for cells in the central nervous system, and disease outcome in the infected host (73). Previous studies using reassortant genetics and comparative sequence analysis demonstrated that these phenotypes segregate most strongly with viral attachment protein 1, suggesting that reovirus serotypes bind to different receptors (74, 76, 77). However, the 1-encoding S1 gene is not the sole determinant of reovirus growth at some sites within the host. For example, the 2-encoding L2 gene influences viral growth in the intestine (11) and spread to new hosts (46). Moreover, JAM-A functions as a receptor for all three reovirus serotypes (16); therefore, JAM-A cannot explain serotype-dependent differences in reovirus pathogenesis. The presence or absence of particular integrins at distinct physiologic sites may critically influence the course of reovirus infection. In support of a role for coreceptor utilization in reovirus growth, reovirus infection can occur in the absence of 1 (19) or JAM-A (5), albeit at greatly reduced efficiency. These findings highlight the complex nature of reovirus attachment and entry and suggest that reovirus tropism and pathogenesis are not dictated by primary receptor interactions alone. It is possible that tropism and pathogenesis are determined by the concerted action of attachment and internalization receptors, perhaps not all of which have been discovered.
ACKNOWLEDGMENTS
We thank Roy Zent for helpful discussions, members of our laboratory for review of the manuscript, Kathy Allen and the Nashville Veterans Affairs Hospital Flow Cytometry Facility for assistance with flow cytometry, the Vanderbilt Imaging Core for help with confocal microscopy, and Aaron Derdowski for assistance with the confocal image analysis. We thank Xuemin Chen and Paul Spearman for providing CEFs, Richard Hynes for providing murine integrin cDNA constructs, Deane Mosher for providing GD25 and GD251A cells, Charles Parkos for providing hJAM-A-specific MAb J10.4 and control ascites, Beat Imof for providing mJAM-A-specific MAb H202-106-7-4, Richard Bankert for providing 2-specific MAb 6F1, and John Williams for providing antibodies.
This research was supported by Public Health Service awards AI007281 (M.S.M.), CA09385 (J.C.F.), AI07474 (S.A.K.-B.), and AI32539, the Vanderbilt University Research Council (J.C.F.), and the Elizabeth B. Lamb Center for Pediatric Research. Additional support was provided by Public Health Service awards CA68485 for the Vanderbilt Cancer Center and DK20593 for the Vanderbilt Diabetes Research and Training Center.
These authors contributed equally to the manuscript.
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Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
The Scripps Research Institute, La Jolla, California 92037
Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
ABSTRACT
Reovirus infection is initiated by interactions between the attachment protein 1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein 2, which serves as the structural base for 1, suggests that integrins mediate reovirus endocytosis. A 1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a 1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in 1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing 1. However, reovirus bound equivalently to cells that differed in levels of 1 expression, suggesting that 1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into 1-deficient cells was substantially diminished in comparison to viral uptake into 1-expressing cells. These data provide evidence that 1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.
INTRODUCTION
Viral attachment and cell entry are key determinants of target cell selection in the infected host and thus play important roles in pathogenesis. Many viruses interact with multiple cell surface molecules to mediate the processes of attachment and internalization (68). For example, human immunodeficiency virus uses CD4 to bind the cell surface and chemokine receptors to facilitate the conformational alterations in envelope glycoproteins that culminate in fusion of the viral envelope and cell membrane (35). Receptors that serve as initial binding sites have been identified for many viruses (25). However, little is known about the postattachment events that lead to nonenveloped virus internalization, in particular those that mediate virus uptake into the endocytic pathway.
Mammalian reoviruses are large, nonenveloped, double-stranded RNA-containing viruses that infect a variety of mammalian species. Following attachment to target cells, reoviruses are internalized by receptor-mediated endocytosis (3, 13, 14, 72), which is mostly likely to be clathrin dependent (31). Proteolytic disassembly in endosomes leads to removal of outer capsid protein 3 and cleavage of outer capsid protein μ1 (3, 14, 27, 72). The resultant disassembly intermediate formed by these events, the infectious subvirion particle (ISVP), is capable of penetrating endosomal membranes in a μ1-dependent manner to release the transcriptionally active viral core particle into the cytoplasm (17, 18, 58), where viral replication takes place. Cellular determinants of reovirus receptor-mediated internalization following attachment and preceding uncoating are poorly defined.
We previously identified junctional adhesion molecule A (JAM-A) as a serotype-independent receptor for reovirus (5, 16, 34). JAM-A is a type 1 transmembrane protein expressed in a variety of cell types, including polarized endothelial and epithelial cells and circulating leukocytes (52, 55, 81). JAM-A interacts with several scaffolding proteins and cytoplasmic adaptor molecules (6, 28, 29) and is hypothesized to play an important role in maintaining the barrier function of epithelial junctions (48, 52, 55, 60). JAM-A is phosphorylated during platelet activation and required for mitogen-activated protein kinase activation following treatment of endothelial cells with basic fibroblast growth factor (57). These data indicate that JAM-A is intimately associated with cytoskeletal and signaling machinery, which raises the possibility that reovirus binding to JAM-A mediates cytoskeletal rearrangement or signaling events to facilitate virus internalization.
The attachment mechanisms of reovirus and adenovirus are remarkably similar (70, 71). The trimeric attachment proteins of both viruses, 1 and fiber, respectively, are structural homologues and fold using a highly unusual triple -spiral motif (10, 20, 83). The globular head domains of these molecules are formed from eight-stranded -barrels with identical interstrand connectivity (70). The receptors for 1 and fiber, JAM-A (5) and coxsackievirus and adenovirus receptor (CAR) (7), respectively, are two-domain, immunoglobulin superfamily proteins that form homodimers using analogous molecular surfaces (71). Also, both JAM-A and CAR localize to tight junctions in polarized epithelial cells (23, 52, 55, 60). Remarkably, reovirus and adenovirus engage their respective receptors by thermodynamically favored disruption of receptor homodimers (34, 53).
Despite mediating high-affinity attachment of adenovirus to cells, engagement of CAR does not permit efficient adenovirus internalization. Instead, adenovirus entry is enhanced by high-avidity interactions of the viral penton base complex with integrins, including v3 and v5 (80). Integrins are heterodimeric cell surface molecules that consist of and subunits (43). Integrins function to mediate cellular adhesion to the extracellular matrix, regulate cellular trafficking, and transduce both outside-in and inside-out signaling events (42). In addition to adenovirus, several other pathogenic microorganisms have usurped the adhesion and signaling properties of integrins to bind or enter host cells (1, 8, 32, 39-41, 44, 49).
To define the molecular basis of reovirus internalization, we first tested the capacity of a JAM-A mutant lacking a cytoplasmic tail to support reovirus attachment and infection. We found that while JAM-A is necessary for efficient attachment to cells, the JAM-A cytoplasmic tail is not required for reovirus infection. Given the mechanistic conservation of reovirus and adenovirus attachment strategies and the observation that reovirus outer capsid protein 2 contains the conserved integrin-binding sequences Arg-Gly-Asp (RGD) and Lys-Gly-Glu (KGE), we tested the role of integrins in reovirus internalization. We found that infection by reovirus virions is inhibited by antibodies specific for 1 integrin. In addition, cells deficient in 1 integrin have a diminished susceptibility to reovirus infection due to a postattachment block to viral entry. Together, these data indicate that, following attachment to JAM-A, 1 integrin facilitates internalization of reovirus into cells. Our findings further demonstrate that two seemingly unrelated viruses utilize distinct cellular molecules to mediate attachment and internalization in a remarkably similar manner.
MATERIALS AND METHODS
Cells, viruses, and antibodies. Spinner-adapted murine L929 (L) cells were grown in either suspension or monolayer cultures in Joklik's modified Eagle's minimal essential medium (Irvine Scientific, Santa Ana, CA) supplemented to contain 5% fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin per ml, 100 U of streptomycin per ml, and 0.25 mg amphotericin per ml (Gibco Invitrogen Corp., Grand Island, NY). Chinese hamster ovary (CHO) cells were maintained in Ham's F12 medium (Irvine Scientific) supplemented to contain 10% fetal bovine serum, 100 U of penicillin per ml, and 100 U of streptomycin per ml. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Gibco Invitrogen Corp.) and supplemented as described for CHO cells. Primary cultures of chicken embryo fibroblasts (CEFs) were obtained from Paul Spearman (Vanderbilt University) and maintained in medium 199 with Earle's salts and 2.2 mg sodium bicarbonate per ml (Gibco Invitrogen Corp.) supplemented to contain 5% fetal bovine serum, 10% tryptose phosphate broth, 1% chicken serum (Gibco Invitrogen Corp.), and antibiotics as described for CHO cells. GD25 and GD251A cells were obtained from Deane Mosher (University of Wisconsin, Madison) (78) and maintained as described for HeLa cells. Medium for GD251A cells was supplemented to contain 10 μg of puromycin (Sigma-Aldrich, St. Louis, MO) per ml to maintain 1 integrin expression.
Reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) are laboratory stocks. Working stocks of virus were prepared by plaque purification and passage in L cells (75). Purified virions were generated from second-passage L-cell lysate virus stocks. Virus was purified from infected cell lysates by Freon extraction and CsCl gradient centrifugation as described (36). Bands corresponding to the density of reovirus particles (1.36 g/cm3) were collected and dialyzed against virion storage buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl [pH 7.4]). Reovirus particle concentration was determined by the equivalence of 1 unit of optical density at 260 nm to 2.1 x 1012 particles (69).
Viral infectivity titers were determined by either plaque assay (75) or fluorescent focus assay (4). ISVPs were generated by treatment of 2 x 1011 virion particles per ml with 2 mg of -chymotrypsin (Sigma-Aldrich) per ml in a volume of 100 μl virion storage buffer at 37°C for 30 min (2). Reactions were terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 1.0 mM. Purified T1L virions in carbonate-bicarbonate buffer (Sigma-Aldrich) were fluoresceinated by incubation with 50 μg fluorescein isothiocyanate (FITC) (Pierce, Rockford, IL) per ml at room temperature for 1 h (38). Excess FITC was removed by exhaustive dialysis against phosphate-buffered saline (PBS).
Immunoglobulin G (IgG) fractions of rabbit antisera raised against T1L and T3D (79) were purified by protein A-Sepharose as previously described (4). Fluorescently conjugated secondary Alexa antibodies were obtained from Molecular Probes (Invitrogen, San Diego, CA). The human JAM-A (hJAM-A)-specific monoclonal antibody (MAb) J10.4 and control mouse ascites were provided by Charles Parkos (Emory University School of Medicine) (52), and the murine JAM-A (mJAM-A)-specific MAb H202-106-7-4 was provided by Beat Imof (University of Geneva). The human 2-specific MAb AA10 (IgM) (8) and human 1-specific MAb DE9 (IgG1) (8) were used as diluted ascites. Human integrin-specific MAbs MAB1980 (v), MAB1973 (1), MAB2057 (3), MAB1378 (6), MAB1976 (v3), and MAB1961Z (v5) were purchased from Chemicon International (Temecula, CA). Antibody BIIG2 (5) (Developmental Hybridoma Studies Bank, University of Iowa, Iowa City, IA) was provided by John Williams (Vanderbilt University). Function-blocking human 2-specific MAb 6F1 was provided by Richard Bankert (State University of New York at Buffalo). Function-blocking murine 1 MAb CD29 (IgM) and hamster IgM isotype control were purchased from BD Biosciences Pharmingen (San Jose, CA). Murine 1-specific MAb MAB1997 (Chemicon) and human 1-specific MAb MAB2253Z (Chemicon) were used to assess expression of 1 integrin on GD25 and GD251A cells and HeLa cells, respectively, by flow cytometry. ICAM-1-specific MAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies used for flow cytometric analysis of HeLa cells are shown in Table 1.
Sequence analysis. The sequences of the reovirus 2-encoding L2 gene from strains T1L (NC_004259), type 2 Jones (T2J) (NC_004260), T3D (NC_004275), T1Neth85 (AF378004), T2SV59 (AF378006), T3C9 (AF378007), T3C18 (AF378008), T3C87 (AF378009), and T3C93 (AF378010) were aligned using the protein sequence alignment algorithm in MacVector, version 8.0.1 (Accelrys, San Diego, CA).
Plasmid constructs. Human JAM-A was subcloned into expression plasmid pcDNA3.1+ (Invitrogen) (34). Truncation mutant JAM-A-CT was generated by PCR using full-length JAM-A cDNA as the template. Amino acids 1 to 260 (261-299) were cloned and appended with a stop codon using T7 primer and 5'-TACGGGATCCTCAGGCAAACCAGATGCC-3' as the forward and reverse primers, respectively. The gene-specific primer encompasses nucleotides 981 to 995 of the JAM-A cDNA. The PCR product was digested with BamHI (recognition site underlined in the reverse primer sequence) and subcloned into the complementary restriction sites of pcDNA3.1+. Fidelity of cloning was confirmed by automated sequencing. Plasmid constructs encoding murine integrin v (33) and 2 (30) were previously described. A cDNA encoding murine 1 integrin cloned into the EcoRI site of pGEM1 was obtained from Richard Hynes (Massachusetts Institute of Technology) (D. W. DeSimone, V. Patel, and R. O. Hynes, unpublished). Integrin cDNAs were subcloned into the expression plasmid pcDNA3.1+.
Transient transfection of CHOs and CEFs. Monolayers of cells in a 24-well plate (Costar, Cambridge, MA) were transfected with empty vector or plasmids encoding receptor constructs by using Lipofectamine Plus reagent (Invitrogen). Cells were incubated for 24 h to allow receptor expression prior to adsorption with either reovirus virions or ISVPs for infectivity studies.
Flow cytometric analysis. Surface expression of integrin subunits on HeLa cells was determined by flow cytometry. Cells were detached from plates by using PBS-EDTA (20 mM EDTA). Cells were washed and centrifuged at 2,000 x g to form a pellet, resuspended with integrin-specific or control antibodies in PBS- bovine serum albumin (BSA) (Sigma-Aldrich) (5% BSA), and incubated at 4°C for 1 h. Cells were washed twice and incubated with an appropriate secondary antibody conjugated to R-phycoerythrin (BD Biosciences Pharmingen) at 4°C for 1 h. Cells were washed, resuspended in PBS, and analyzed by flow cytometry. Results were analyzed using the Windows Multiple Document 2.8 flow cytometry application (The Scripps Research Institute, La Jolla, CA).
The mean fluorescence intensity was measured for an average of 14,000 gated events for cells treated with control or integrin-specific antibodies. Events were gated relative to cells stained with an appropriate secondary antibody conjugated to phycoerythrin. Reovirus binding to GD25 and GD251A cells was analyzed by adsorbing cells with 2 x 1011 FITC-labeled particles of strain T1L at 4°C for 1 h. Cells were washed and analyzed by flow cytometry.
Fluorescent focus assay of viral infection. Cells were plated in 24-well or 96-well plates (Costar) and adsorbed with virus at various multiplicities of infection (MOIs) at either 4°C or room temperature for 30 to 60 min. Inocula were removed, cells were washed, and complete medium was added. Infected cells were incubated at 37°C for 16 to 24 h to allow a single cycle of viral replication. Cells were fixed with methanol at –20°C for at least 30 min. Fixed cells were incubated with PBS-BSA (5% BSA) for at least 15 min, followed by incubation with reovirus-specific polyclonal antiserum (1:500) in PBS-Triton X-100 (0.5% Triton X-100) at room temperature for 1 h. Cells were washed twice and incubated with an Alexa 488- or 546-labeled anti-rabbit IgG (1:1,000) in PBS-Triton X-100 (0.5% Triton X-100) at room temperature for 1 h.
Cells were washed twice and visualized by indirect immunofluorescence at a magnification of 20x using an Axiovert 200 fluorescence microscope (Carl Zeiss, New York, NY). Infected cells (fluorescent focus units [FFU]) were identified by diffuse cytoplasmic fluorescence staining that was excluded from the nucleus. Reovirus-infected cells were quantified by counting random fields of view of equivalently confluent monolayers for three to five fields of view for triplicate wells or by counting the entire well for triplicate wells (4).
Confocal imaging of reovirus internalization. GD25 and GD251A cells were plated on coverslips in 24-well plates. Cells were chilled at 4°C for 45 min prior to infection, washed with PBS, adsorbed with 8 x 105 particles per cell of T1L virions in gelatin saline, and returned to 4°C for 1 h. The MOI used was the minimum number of particles required to detect signal by confocal immunofluorescence microscopy at early time points postinfection. Cells were either washed and fixed or nonadherent reovirus was aspirated and replaced with warm Dulbecco's modified Eagle's medium and returned to 37°C. At 10-min intervals, cells were washed with PBS and fixed with 4% formaldehyde for 20 min. Excess formaldehyde was quenched with an equal amount of 0.1 M glycine, followed by washing with PBS. Cells were treated with 1% Triton X-100 for 5 min and incubated with PBS-BGT (PBS, 0.5% BSA, 0.1% glycine, and 0.05% Tween 20) for 10 min. Cells were incubated with reovirus-specific polyclonal antiserum (1:500) in PBS-BGT for 1 h and washed with PBS-BGT. Cells were stained with donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 488 (Molecular Probes) (1:500) to visualize reovirus, phalloidin conjugated to Alexa Fluor 546 (Molecular Probes) (1:100) to visualize actin, and TO-PRO 3 conjugated to Alexa Fluor 642 (Molecular Probes) (1:1,000) to visualize DNA. Cells were incubated for 1 h with secondary antibodies and fluorescent probes in PBS-BGT and washed with PBS-BGT. Coverslips were removed from wells and placed on slides using Prolong Anti-Fade mounting medium (Molecular Probes). Images were captured on a Zeiss LSM 510 laser-scanning confocal microscope using LSM 510 software.
Virus internalization was quantified by enumerating fluorescent particles localized at the cell periphery and particles internalized into the cytoplasm to determine the total number of fluorescent particles per cell. Ten cells were analyzed for each time point. The number of internalized particles was measured as a percentage of the total number of particles per cell.
Statistical analysis. Means of triplicate samples were compared by using an unpaired Students' t test (Microsoft Excel, Redmond, WA). P values of <0.05 were considered statistically significant.
RESULTS
The JAM-A cytoplasmic tail is dispensable for reovirus infection. JAM-A is a serotype-independent reovirus receptor with a cytoplasmic tail known to interact with a variety of proteins (6, 28, 29). To determine whether the JAM-A cytoplasmic tail is required for reovirus entry, we generated a JAM-A cytoplasmic tail deletion mutant (JAM-A-CT) and tested its capacity to support reovirus infection following transfection of CHO cells. CHO cells do not express detectable levels of JAM-A (55, 60) and are poorly permissive for reovirus infection (34). Cells were transiently transfected with plasmids encoding full-length JAM-A, JAM-A-CT, or empty vector as a control. Equivalent cell surface expression of transfected constructs was confirmed by flow cytometry (data not shown).
The capacity of reovirus to infect CHO cells following transfection with the JAM-A constructs was tested using reovirus fluorescent focus assays. Following transient transfection of CHO cells with empty vector, JAM-A, or JAM-A-CT, cells were adsorbed with reovirus strains T1L and T3D and scored for infection by indirect immunofluorescence at 20 h postinfection (Fig. 1). Expression of either full-length or truncated JAM-A was sufficient to allow reovirus infection of CHO cells, permitting viral protein production of both type 1 and type 3 reovirus strains. These results indicate that the JAM-A cytoplasmic tail is not required for efficient reovirus attachment and infection.
Reovirus outer capsid proteins contain integrin-binding sequences. Structural and functional analyses indicate that reovirus and adenovirus share remarkably similar mechanisms of attachment (70, 71). To determine whether reovirus outer capsid proteins contain sequences that could potentially engage integrins, we performed a search for integrin-binding motifs in the 1, 3, μ1, and 2 proteins, which form the reovirus outer capsid (26). We identified two common integrin-binding motifs, RGD and KGE, in the deduced amino acid sequence of the 2 protein (Fig. 2). The RGD motif is conserved in all reovirus strains for which sequence information is available (15, 67); the KGE motif is conserved in all of those strains except T2J (15, 67). The 2 protein is a component of the reovirus outer capsid and core (26). It is structurally arranged as a pentamer at the virion fivefold axes of symmetry and forms the base for attachment protein 1 (26, 63). The presence of conserved integrin-binding motifs in the reovirus 2 protein led us to test whether reovirus utilizes integrins to mediate internalization.
An antibody specific for 1 integrin inhibits reovirus infection of HeLa cells. To determine whether integrins are required for reovirus infection, we first used flow cytometry to analyze integrin expression on the surface of HeLa cells. HeLa cells were incubated with integrin-specific MAbs and a phycoerythrin-labeled secondary antibody (Table 1). RGD-binding integrin subunits 3, 5, v, and 1 and KGE-binding integrin subunits 1, 2, 6, and 1 (43) were detected on HeLa cells at levels above those in control antibody-treated cells. RGD-binding integrin heterodimer v5 also was detected at levels above that of the control, while there was low-level expression of v3. Thus, HeLa cells express both RGD- and KGE-binding integrins.
To assess a role for integrins in reovirus replication, we tested antibodies specific for the RGD- and KGE-binding integrins expressed on HeLa cells for the capacity to block reovirus infection. HeLa cells were incubated with integrin-specific and control antibodies prior to adsorption with reovirus virions. Viral infection was detected by indirect immunofluorescence (Fig. 3A). We found that 1-specific MAb DE9 resulted in a 50% reduction in infection (P < 0.05), while antibodies specific for the other integrin subunits expressed on HeLa cells had no effect. Control antibodies produced anticipated effects; JAM-A-specific MAb J10.4 inhibited infection, whereas ICAM-specific MAb (data not shown) or control mouse ascites (Fig. 3A) did not. The effect of MAb DE9 was dose dependent (Fig. 3B), providing further evidence that the inhibition of infection was dependent on integrin blockade.
To determine whether particular subunits pair with 1 integrin to facilitate reovirus infection, we tested whether treatment with integrin-specific antibodies was capable of enhancing the inhibitory effect of 1 integrin-specific MAb DE9 on reovirus infection. We also tested whether antibodies specific for other integrin subunits expressed on HeLa cells, 3 and 5, were capable of infection blockade. HeLa cells were treated with MAb DE9 in combination with other integrin-specific antibodies prior to adsorption with reovirus virions (Fig. 3C). While treatment of HeLa cells with MAb DE9 resulted in a 50% reduction in reovirus infection, none of the other integrin-specific antibodies tested reduced reovirus infection to a greater extent than that resulting from treatment with DE9 alone. These results suggest that the integrin epitope bound by reovirus is blocked by 1-specific MAb DE9 and not by the other MAbs used in these experiments.
JAM-A MAb J10.4 blocks reovirus infection 90% (Fig. 3A). To determine whether the residual level of infection in the presence of MAb J10.4 was dependent on reovirus interactions with 1 integrin, we treated HeLa cells with JAM-A-specific MAb J10.4 in combination with MAb DE9 prior to adsorption with reovirus virions (Fig. 3D). Treatment of HeLa cells with MAb J10.4 and MAb DE9 completely abrogated reovirus infection, indicating that the effect of JAM-A blockade is enhanced when 1 integrin is not available for interactions with reovirus. Treatment with MAb DE9 did not significantly inhibit infection by ISVPs (Fig. 3D), suggesting that viral attachment is not affected by 1 integrin blockade. Taken together, these results support the conclusion that a 1-specific antibody blocks reovirus infection at a step subsequent to attachment but prior to uncoating, implicating 1 integrin in reovirus internalization.
Transient transfection of integrin cDNAs allows reovirus infection of JAM-A-expressing CEFs. Ectopic expression of JAM-A in CEFs rescues infection by reovirus ISVPs but not by virions (5), suggesting that these cells exhibit a cell-specific block at the entry or uncoating phases of reovirus infection. To test the capacity of integrins to confer infection of CEFs by reovirus virions, CEFs were transiently transfected with a JAM-A-encoding plasmid in the presence or absence of murine v, 2, or 1 integrin-encoding plasmids singly or in pairs. Transfected cells were infected with reovirus virions or ISVPs, and infection was assessed by indirect immunofluorescence (Fig. 4). Expression of 1 integrin paired with either of the murine integrin subunits provided an approximately fourfold enhancement of infection by reovirus virions in comparison to that in cells transfected with JAM-A alone. These data suggest that 1 integrin expression complements a reovirus cell entry defect in CEFs and provide further support for the involvement of 1 integrin in reovirus internalization.
Cells deficient in 1 integrin have a decreased capacity to support reovirus infection. To further assess a role for 1 integrin in reovirus infection, we tested the capacity of reovirus to infect cells deficient in the 1-integrin subunit. GD25 cells are murine embryonic stem cells derived from 1-null embryos (78). GD251A cells are GD25 cells that have been engineered to stably express 1 integrin and thus serve as an isogenic control for GD25 cells. Flow cytometric analysis confirmed that while both cells express JAM-A, only GD251A cells express 1 integrin (Fig. 5A). GD25 cells (1–/–) and GD251A cells (1+/+) (78) were adsorbed with reovirus virions or ISVPs, and infection was scored by indirect immunofluorescence (Fig. 5B). In comparison to 1+/+ cells, 1–/– cells were substantially less susceptible to infection by virions, while infection by ISVPs was equivalent in both cell types. Importantly, preincubation of 1+/+ cells with murine 1 integrin-specific MAb CD29 reduced infection in 1+/+ cells (Fig. 5C), indicating that enhancement of infection is due to expression of 1 integrin. Therefore, 1 integrin is required for efficient reovirus infection.
Reovirus binding to 1–/– and 1+/+ cells is equivalent. Equivalent infection of 1–/– and 1+/+ cells by ISVPs (Fig. 5B) suggests that reovirus is capable of efficiently binding to both cell types. To directly test this hypothesis, 1–/– and 1+/+ cells were mock treated or incubated with FITC-labeled virions and binding was assessed by flow cytometry (Fig. 6). In these experiments, we found that reovirus binds equivalently to 1–/– and 1+/+ cells. These data demonstrate a function for 1 integrin in reovirus infection at a step subsequent to viral attachment.
1 integrin enhances the efficiency of reovirus internalization. To directly assess the role of 1 integrin in reovirus internalization, 1–/– and 1+/+ cells were infected at 4°C and then warmed to 37°C over a time course concurrent with reovirus entry (2, 72). At 10-min intervals, cells were fixed, stained for indirect immunofluorescence, and examined by confocal microscopy. Representative confocal micrographic images of reovirus-infected 1–/– and 1+/+ cells are shown in Fig. 7. Immediately after viral adsorption, both cell types exhibited reovirus staining at the cell periphery. At 10 min postadsorption, some reovirus staining was observed at the cell periphery, yet intracellular staining in 1+/+ cells was also observed. At 20 and 30 min postadsorption, the majority of virions had entered the 1+/+ cells and had a perinuclear location. In sharp contrast to the findings made using 1+/+ cells, viral entry was markedly delayed in 1–/– cells, with the majority of reovirus virions remaining at the cell periphery throughout the time course. At later time points (30 min postadsorption), some virions were present within the cytoplasm, but these were the minority. These findings suggest that expression of 1 integrin enhances reovirus entry.
To quantify reovirus internalization into 1–/– and 1+/+ cells, we determined the number of internalized fluorescent particles as a percentage of the total number of fluorescent particles per cell at various times postadsorption (Fig. 8). At 0 and 10 min postadsorption, the percentage of particles internalized into 1–/– and 1+/+ cells was equivalent, 10 and 30%, respectively. However, at 20 and 30 min postadsorption, the percentage of reovirus particles internalized into 1+/+ cells was 50%, while the percentage of particles internalized into 1–/– cells was only 30% (P < 0.05) (Fig. 8). These data indicate that 1 integrin enhances reovirus entry at early times postadsorption, suggesting a direct role for 1 integrin as a reovirus internalization receptor.
DISCUSSION
In this study, we performed experiments to define the molecular determinants of reovirus internalization. We show that antibodies specific for 1 integrin inhibit reovirus infection at a postattachment step. We provide evidence that expression of 1 integrin promotes infection by reovirus virions in cells with a block to viral internalization and that viral entry is substantially diminished in cells deficient in 1 integrin expression. Together, these data provide strong evidence that 1 integrin serves as a coreceptor to mediate reovirus internalization. These findings suggest a new model for attachment and cell entry of reovirus (Fig. 9). In this model, we propose that reovirus initially interacts with cells via low-affinity binding to carbohydrate. These interactions are followed by high-affinity engagement of JAM-A, which positions the virus on the cell surface for subsequent interactions with 1 integrin to trigger viral endocytosis.
Integrins have been identified as attachment and entry receptors for several viruses, including echovirus (21) (8), foot-and-mouth disease virus (v1, v3, and v6) (9, 44, 45), hantaviruses NY-1 and Sin Nombre virus (3 integrins) (37), Kaposi sarcoma herpesvirus (31) (1), and cytomegalovirus (21, 61, and v3) (32). The Reoviridae family member rotavirus also engages a variety of integrins for attachment and cell entry. Rotavirus strains RRV, SA11, and Wa bind to the I (inserted) domain of 21 integrin via an Asp-Gly-Glu integrin-binding motif in the VP4 spike protein to effect viral attachment (39). The interactions of rotavirus outer capsid protein VP7 with integrins x2 and v3 can mediate viral entry (39, 82). Integrin 41 also can serve as a receptor for rotavirus strain SA11, which contains 41 integrin-binding sequences Leu-Asp-Val in VP7 and Ile-Asp-Ala in VP4 (41). Interestingly, like reovirus, adenovirus engages a specific cell surface protein, CAR, prior to interactions with integrins, which function subsequent to viral attachment to mediate viral endocytosis (50, 80). Therefore, the identification of 1 integrin as a reovirus internalization receptor suggests that the conservation of attachment strategies used by reovirus and adenovirus (70, 71) extends to mechanisms of internalization.
Although the specific reovirus protein required for integrin binding is not apparent from our studies, the 2 protein is a promising candidate. The 2 protein forms a pentameric turret at the virion fivefold symmetry axes and serves as the insertion site for trimers of attachment protein 1 (26). Thus, 2 is the reovirus analogue of the adenovirus penton base protein, which mediates the engagement of integrins by adenovirus (22, 80). Interestingly, 2 also contains conserved RGD and KGE motifs (15), the preferred interaction motifs for several 1 integrin heterodimers (43).
Structural information for 2 is available in the context of the reovirus core but not for the intact virion. In the core, the KGE motif is exposed on the top of the 2 turret, where it would be accessible to a receptor. The RGD motif is also surface exposed, but it appears to be less accessible. However, the 2 structure in the core may not be identical to that in the virion, as the protein undergoes major conformational changes during virion-to-core disassembly (26). Therefore, it is possible that both the RGD and KGE motifs are accessible to interactions with 1 integrin during engagement of the cell surface by the virus.
A human 1 integrin-specific antibody (DE9) reduced reovirus infection of HeLa cells by 50% (Fig. 3). Similarly, a murine 1 integrin-specific antibody (CD29) blocked infection of 1-expressing mouse embryonic stem cells by 50% (Fig. 4). Interestingly, MAb DE9 also blocks infection of echovirus (8) and cytomegalovirus (32), suggesting that an epitope in 1 integrin recognized by MAb DE9 may be a preferred binding site for multiple viruses. It is possible that the residual level of reovirus infection following 1 integrin antibody treatment is attributable to other internalization receptors on the cell surface that may be integrin or nonintegrin molecules. However, it is noteworthy that treatment of HeLa cells with both MAb DE9 and JAM-A-specific MAb J10.4 completely abolishes reovirus growth (Fig. 3D). This finding suggests that the residual infection in J10.4-treated HeLa cells is due to reovirus interactions with 1 integrin. Thus, it appears that blockade of reovirus infection by integrin-specific antibodies is inefficient because complete inhibition of virus-integrin interactions is not possible if the virus is tightly adhered to the cell surface by JAM-A.
Since antibodies specific for 3 and 5 integrins did not inhibit reovirus infection, it is likely that only 1 integrin can serve a reovirus internalization function. Antibodies specific for the integrin subunits expressed on HeLa cells did not further reduce reovirus infection following treatment with a 1 integrin-specific antibody (Fig. 3C). We envision three possible explanations for this result. First, reovirus may directly engage a ligand-binding domain of 1 integrin. Second, reovirus may utilize 1 integrin when paired with numerous subunits that have redundant functions. However, treatment of HeLa cells with a 1 integrin-specific antibody and a mixture of antibodies specific for 1, 2, 3, 5, 6, and v integrins did not diminish reovirus infection in comparison to cells treated with a 1 integrin-specific antibody alone (data not shown). Third, reovirus may engage an epitope of an integrin subunit that is not recognized by the antibodies used in our experiments. Further studies are required to define the biophysical basis of reovirus-integrin interactions.
JAM-A is required for high-affinity reovirus attachment to numerous cell types (5, 16, 34, 56). However, the JAM-A cytoplasmic tail is not necessary for viral endocytosis (Fig. 1). JAM-A likely tethers the virus to the cell surface to facilitate secondary interactions with 1 integrin (Fig. 9). This model is analogous to the mechanism of lymphocyte homing, in which adhesion molecules such as JAM-A provide initial cellular contacts to facilitate subsequent interactions with integrins for diapedesis or signaling (65). An interesting possibility is that JAM-A may be associated with 1 integrin on the host cell plasma membrane. If such were the case, initial JAM-A engagement might facilitate integrin binding, clustering, and viral endocytosis. In support of this hypothesis, JAM-A has been shown to regulate 1 integrin expression and localization (54).
The cytoplasmic domains of integrin subunits are involved in a number of signaling pathways (42). The 1 integrin cytoplasmic domain is linked to cytoskeletal proteins, including talin (62) and -actinin (59), and signaling molecules, including paxillin and focal adhesion kinase (66). In addition, the 1 integrin cytoplasmic domain contains two Asn-Pro-any residue-Tyr (NPXY) motifs (64), which are common sequence motifs in the cytoplasmic domains of many receptors and serve as recognition sites for the cellular endocytic machinery (21, 24). NPXY motifs interact with the μ2 subunit of the adaptor protein 2 complex (12, 61), which can recruit clathrin and trigger clathrin-mediated endocytosis (47).
Since clathrin-dependent mechanisms have been implicated in reovirus cell entry (31), it seems plausible that reovirus engagement of 1 integrin leads to clathrin-mediated endocytosis through signaling regulated by the 1 integrin cytoplasmic domain. It is noteworthy that Kaposi's sarcoma-related herpesvirus binding to 31 integrin (1) and cytomegalovirus binding to 1 integrin (32) activate focal adhesion kinase. In addition, adenovirus engagement of v integrins induces activation of phosphoinositide-3-OH kinase, which is required for adenovirus endocytosis (51).
Identification of 1 integrin as a receptor that triggers reovirus entry raises the possibility that coreceptor binding influences reovirus tropism and disease. Reovirus serotypes differ in mechanisms of spread, tropism for cells in the central nervous system, and disease outcome in the infected host (73). Previous studies using reassortant genetics and comparative sequence analysis demonstrated that these phenotypes segregate most strongly with viral attachment protein 1, suggesting that reovirus serotypes bind to different receptors (74, 76, 77). However, the 1-encoding S1 gene is not the sole determinant of reovirus growth at some sites within the host. For example, the 2-encoding L2 gene influences viral growth in the intestine (11) and spread to new hosts (46). Moreover, JAM-A functions as a receptor for all three reovirus serotypes (16); therefore, JAM-A cannot explain serotype-dependent differences in reovirus pathogenesis. The presence or absence of particular integrins at distinct physiologic sites may critically influence the course of reovirus infection. In support of a role for coreceptor utilization in reovirus growth, reovirus infection can occur in the absence of 1 (19) or JAM-A (5), albeit at greatly reduced efficiency. These findings highlight the complex nature of reovirus attachment and entry and suggest that reovirus tropism and pathogenesis are not dictated by primary receptor interactions alone. It is possible that tropism and pathogenesis are determined by the concerted action of attachment and internalization receptors, perhaps not all of which have been discovered.
ACKNOWLEDGMENTS
We thank Roy Zent for helpful discussions, members of our laboratory for review of the manuscript, Kathy Allen and the Nashville Veterans Affairs Hospital Flow Cytometry Facility for assistance with flow cytometry, the Vanderbilt Imaging Core for help with confocal microscopy, and Aaron Derdowski for assistance with the confocal image analysis. We thank Xuemin Chen and Paul Spearman for providing CEFs, Richard Hynes for providing murine integrin cDNA constructs, Deane Mosher for providing GD25 and GD251A cells, Charles Parkos for providing hJAM-A-specific MAb J10.4 and control ascites, Beat Imof for providing mJAM-A-specific MAb H202-106-7-4, Richard Bankert for providing 2-specific MAb 6F1, and John Williams for providing antibodies.
This research was supported by Public Health Service awards AI007281 (M.S.M.), CA09385 (J.C.F.), AI07474 (S.A.K.-B.), and AI32539, the Vanderbilt University Research Council (J.C.F.), and the Elizabeth B. Lamb Center for Pediatric Research. Additional support was provided by Public Health Service awards CA68485 for the Vanderbilt Cancer Center and DK20593 for the Vanderbilt Diabetes Research and Training Center.
These authors contributed equally to the manuscript.
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