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Cell integrins: commonly used receptors for diverse viral pathogens
Review
TRENDS in Microbiology
Vol.15 No.11
Cell integrins: commonly used receptors for diverse viral pathogens Phoebe L. Stewart1 and Glen R. Nemerow2 1 2
Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
When searching for their favorite host tissues, animal viruses frequently attach to cell-surface receptors that have key roles in normal cell physiology. Integrins are prime examples of physiologically important receptors that have been usurped by nonenveloped and enveloped viruses for attachment and/or cell entry. This family of heterodimeric receptors mediates cell adhesion, cell migration, tumor metastasis and cell differentiation. Recent investigations have shed new light on integrin structure as well as on the underlying molecular features of their association with viral ligands. In this review, we discuss several examples of virus–integrin interactions that highlight recent advances in this field. The continuing improvements in virus and cell imaging techniques have helped to uncover the molecular basis of how integrins are recognized by such a wide range of microbial pathogens to invade host cells. Integrin structure Integrins are integral membrane proteins, and all a and b subunits contain a single transmembrane spanning helix [1]. Many, but not all, integrins recognize RGD sequences displayed on the exposed loops of viral capsid proteins or extracellular matrix (ECM) proteins [2–6]. Virus or ECM binding induces clustering and/or conformational changes in integrin quaternary structure (for excellent recent reviews, see Refs [7,8]). Conformational changes to integrin can elicit cell-signaling events that increase ligand affinity/avidity as well as promote cytoskeletal rearrangement and virus internalization. As membrane proteins, integrins present significant challenges for structural biology. In addition, integrins undergo conformational changes to achieve outside–in and inside–out signaling, which adds an additional layer of complexity. Although there is still controversy, particularly regarding the extent of conformational change [7,8], structural information has been obtained by a variety of techniques, including X-ray crystallography, NMR, electron microscopy (EM) and three-dimensional image reconstruction of negatively stained and cryopreserved samples. The extracellular portion, or ectodomain, of a and b subunits typically contains 700–1000 amino acids. Alpha subunits contain four or five extracellular domains and a small cytoplasmic domain (Figure 1a). Half of the 18 different a subunits (aD, aE, aL, aM, aX, a1, a2, a10, a11) contain an I domain, also Corresponding author: Nemerow, G.R. ([email protected]). Available online 7 November 2007. www.sciencedirect.com
referred to as a von Willebrand factor A domain, or simply an A domain. When an I domain is present in an a subunit, it is nearly always the ligand-binding site. When the I domain is absent from the a subunit (as in a3, a4, a5, a6, a7, a8, a9, av and aIIb), then the ligand-binding site is typically formed by the b-propeller domain of the a subunit together with an I domain of the b subunit. Beta subunits (b1–b8) contain eight extracellular domains, including an I domain, and a cytoplasmic domain of various lengths (Figure 1b). There are several crystal structures of the a subunit I domain both with and without bound ligands, such as a triple-helical collagen peptide and fragments of intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 [7]. The a I domain has a conserved metal-ion-dependent adhesion site (MIDAS) that binds Mg2+ under physiological conditions, and the residues that coordinate the metal and surround the metal-binding site are important for ligand binding. The conformational changes of the a I domain that occur upon ligand binding involve the shift of an a-helix (a7) away from the ligand-binding site [7]. The b subunit I domain has a similar structure with three metal binding sites: MIDAS, adjacent MIDAS (ADMIDAS) and ligandinduced metal-binding site (LIMBS) [7]. The crystal structure of the extracellular portion, or ectodomain, of avb3 provides a detailed picture of an integrin without an a I domain and reveals a bent conformation for both the a and b subunits [9,10] (Figure 1c). Some studies suggest that a large conformational change, involving straightening of the integrin ectodomain, accompanies RGD ligand binding (the ‘switchblade’ model), whereas other studies suggest a more subtle conformational change without extension (the ‘deadbolt’ model) [7,8]. There is a consensus, however, that some conformational change must occur upon ligand binding to convey a signal from ‘outside–in’. The cytoplasmic domains of the a and b subunits interact with a variety of intracellular proteins, such as kinases and cytoskeletal proteins to promote signaling. The cytoplasmic domains of a and b subunits are all relatively short (20–50 amino acids) with the exception of b4, which has a long cytoplasmic domain of 1000 amino acids and contains four fibronectin type III repeat domains [11]. NMR studies have provided structures of several a subunit and b subunit cytoplasmic tails, as well as the complex of full-length aIIb and b3 cytoplasmic tails [12]. The complex structure shows multiple hydrophobic and electrostatic contacts within the membrane-proximal
0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.10.001
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Figure 1. Integrin domains and heterodimer structure. (a) The structural domains of a subunits with four or five extracellular domains, a transmembrane spanning region and a small cytoplasmic domain. The I domain (magenta) is present only in nine of the 18 different a subunits. The N-terminus is near the b-propeller domain and the C-terminus is cytoplasmic. (b) The structural domains of b subunits with eight extracellular domains, a transmembrane spanning region and cytoplasmic domain. The EGF-1 and EGF-2 domains (gray) are models based on the EGF-3 domain structure in the avb3 crystal structure [10]. The N-terminus is near the PSI domain and the C-terminus is cytoplasmic. The extended domain representations of the a and b subunits shown in (a) and (b) are based on figures in Refs [7,8]. (c) Composite integrin heterodimer structure (bent conformation) based on the crystal structure of the a2 I domain (PDB ID 1AOX) [54], the crystal structure of the avb3 ectodomain (PDB ID 1U8C) [10] and the NMR structure of the cytoplasmic tails of aIIbb3 (PDB ID 1M8O) [12]. The a subunit is shown as mostly blue, with an associated I domain in magenta. The b subunit is shown as red. The EGF-1 and EGF-2 domains, which are missing from the avb3 structure, are not shown. The position of the a I domain is approximate and its effect on the overall fold of the ectodomain is assumed to be minimal for this composite structural model. The orientation of the extracellular domains relative to the membrane is as modeled in an EM study of the avb3 ectodomain complexed with four domains of fibronectin [63]. The membrane is represented by a gray bar, 30 A˚ thick on the molecular scale. The domains are shown as 10 A˚ resolution filtered density maps based on their atomic coordinates.
helices of the a and b cytoplasmic tails. A recent structural study has shed new light on the structural basis of ‘inside– out’ signaling. The intracellular protein talin, which binds to b1, b2 and b3 integrins, connects integrins to the actin cytoskeleton and can alter the affinity of integrins for extracellular ligands. The NMR structure of a talin–integrin complex, combined with analysis of targeted mutations, reveals that the contact between a subdomain of talin and the membrane-proximal region of the b subunit tail is crucial for activation of integrin [13]. Interaction of adenovirus with av integrins Adenovirus was one of the first examples of a virus that was shown to use multiple cell receptors to infect host cells. After attachment of adenovirus to cells via association with coxsackie adenovirus receptor (CAR), various adenovirus serotypes utilize av integrins and b1 integrins for cell entry [4,14–18]. Adenoviruses interact with av integrins via a long flexible RGD loop on the surface of the penton base. The structures of adenovirus types 2 and 12 in complex with a soluble form of avb5 integrin have been determined ˚ ) resolution by cryoelectron microscopy to moderate (21 A (cryoEM) [19]. These structures reveal a ring of integrin density over the five RGD loops of the penton base. Although these cryoEM structures have imposed icosahedral symmetry and represent the average of datasets with 200–500 particle images, they do suggest that multiple integrins can bind at each penton base. This concept was supported by a kinetic analysis (BIAcore) of interactions of adenovirus 2 with avb5 that indicated that four or five integrin molecules bound per penton base at close to saturation. Adenovirus is capable of escaping neutralization by antibodies directed to the RGD sites [20]. This is probably attributable to the shielding effect of the www.sciencedirect.com
adenovirus fiber, which protrudes from the penton base. These structural analyses provided insight into the role of integrin clustering in adenovirus entry. In particular, they led to studies of the relationship between integrin signaling and virus entry and infection (Box 1). Herpesviruses exploit integrins for cell entry and infection In a somewhat surprising development, several large enveloped DNA viruses have been shown to use integrins to invade host cells. Human cytomegalovirus (HCMV), a b-herpesvirus and the most frequent cause of congenital birth defects, initially binds to host cells via association with cell-surface heparan sulfate proteoglycans [21,22]. However, this event is not sufficient to allow HCMV entry, and thus further investigations have sought to identify other receptors that mediate cell entry. Among the many outer envelope glycoproteins of human herpesviruses, one of these, designated gB, is required for subsequent cell entry/fusion [23]. Because HCMV infection is associated with cell signaling events [24] characteristic of integrin ligation, Feire and colleagues [25] searched the sequence of HCMV gB for conventional integrin-binding motifs (i.e. RGD, LDV, DGE) but did not find these sequences. Instead, they noted that gB contains the RX6–8DLXXF consensus sequence present in the ADAM (a disintegrin and a metalloprotease) family of proteins, which recognize integrins, particularly b1 subtypes [26]. This so-called disintegrin sequence is preserved in the gB glycoproteins of many clinical and laboratory strains of HCMV as well as in other g- and b-herpesviruses but not in a- herpesviruses. Using integrin-function-blocking antibodies, peptide antagonists and integrin-deficient cell lines, Feire and co-workers demonstrated that several b1 as well as
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Box 1. Integrins as transducers of cell signaling during virus entry Integrins are heterodimeric cell-surface glycoproteins comprising an a and b subunit. Their association with extracellular matrix proteins or viral capsid proteins can initiate multiple signaling events that facilitate receptor-mediated endocytosis of virus particles. The cytoplasmic domains of b1 and b3 integrin subunits, in particular, associate with several host-cell signaling molecules upon binding to the RGD motifs displayed on the surface of viral ligands. Adenovirus provided one of the first examples of signaling events via ligation of integrin [68]. The association of the adenovirus penton base with av integrins induces receptor clustering, which leads to the tyrosine phosphorylation/activation of focal adhesion kinase (FAK). FAK is well known for its role in cell motility as well as for its impact on downstream molecules such as ERK1/ERK2 mitogen-activated protein kinases (MAP kinases). Interestingly, adenovirus signaling to FAK does not result in enhanced virus entry into cells. Instead, FAK activation might contribute to the production of proinflammatory cytokines [69], a situation that limits the use of adenovirus vectors for systemic delivery in humans. Adenovirus-mediated ligation of integrin also promotes activation of p130CAS, PI3K and the Rho family of small GTPases. One consequence of these signaling events is actin polymerization and enhanced adenovirus internalization. In addition to enhanced endocytosis, PI3K activation might result in increased survival of certain host cells through subsequent signaling to protein kinase B (AKT). This might allow completion of the virus replication cycle and production of progeny virions [70]. Thus, viruses such as adenovirus elicit potent signaling events via ligation of integrin that enhance infection of different host cells. These same signaling events can also affect host immune responses as well as cell survival and cell proliferation.
avb3 integrins engage HCMV gB through its disintegrin motif, thereby facilitating cell entry and infection [25]. Moreover, engagement of integrins by HCMV leads to phosphorylation of the cell-signaling molecule, focal adhesion kinase (FAK) as well as polymerization of the actin cytoskeleton, similar to other integrin-binding viruses, such as adenovirus and Kaposi’s sarcoma-associated herpesvirus (KSHV). Wang and colleagues have also provided evidence for the use of integrin avb3 by HCMV in an RGD-independent manner [27]. Interestingly, it is the gH envelope protein of HCMV, rather than the gB protein, that recognizes integrin avb3 in an RGD-dependent manner. This situation has also been reported to occur with herpes simplex virus type 1 (HSV-1) association with avb3 integrin [28]. Further analyses are needed to uncover the precise structural interactions of HMCV gB with its integrin partners as well as to determine how integrin engagement affects HCMV pathogenesis. Another member of the herpesvirus family, HHV-8, is frequently associated with Kaposi’s sarcoma in patients with HIV/AIDS and is referred to as KSHV. As is the case for most herpesviruses, KSHV uses heparan sulfate for initial cell adhesion [29], whereas entry into cells via endocytosis [30] requires interaction of KSHV gB with integrin a3b1 [31]. Interestingly, Akula and colleagues [31] suggested that a region of the KSHV containing an RGD sequence was important for integrin association on the basis of competition studies with RGD peptides and anti-RGD antibodies. Since these reagents only partially inhibited KSHV infection, it is possible that the disintegrin motif that is also present in KSHV gB [25] contributes to integrin associations. Regardless of the precise mode of www.sciencedirect.com
engagement of integrin, KSHV induces activation of the cell-signaling molecules, RhoA GTPase and Src [32] as well as FAK [33], that promote virus entry and infection. An intriguing question arising from these findings is whether KSHV/integrin-mediated cell signaling might have important consequences for viral pathogenesis. In particular, integrin-mediated cell signaling events could promote the secretion of growth factors and/or cytokines that facilitate epithelial cell proliferation, a situation that could accelerate Kaposi sarcoma tumor formation. In support of this possibility, ligation of integrin a2b1 by certain ECM proteins allows re-epithelization of human skin wounds, a process that requires cell migration and/or proliferation [34]. Thus, whether engagement of related b1 integrins, including a3b1, by KSHV has a significant role in the development of Kaposi sarcoma remains to be determined. Recently, Kaleeba and Berger reported that KSHV uses a distinct receptor for cell membrane fusion after integrinmediated virus uptake [35]. They demonstrated that a 501 amino acid membrane protein known as xCT (the 12transmembrane light chain of the human cysteine–glutamate exchange transporter system, Xc) promotes KSHV membrane fusion with host cells, an event required for successful virus infection. Thus, cells originally resistant to membrane fusion and infection by KSHV could be rendered susceptible by transient expression of xCT. Interestingly, members of the xCT family have been shown to be part of a heterodimeric complex including the 80 kDa CD98 (4F2 antigen) and a 45 kDa light chain including xCT that is capable of regulating b1 integrin signaling [36]. These studies illustrate the interplay between integrins and their associated partners in transmitting cell signals that promote distinct steps in viral invasion. b3 Integrins serve as receptors of pathogenic hantaviruses Hantaviruses are enveloped viruses containing a negative-stranded RNA genome. Several members of this virus family, such as Sin Nombre virus (SNV), cause two highly lethal diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS). Hantaviral infection is associated with disruption of endothelial and platelet functions, which result in clotting defects and acute pulmonary edema. Using competition assays with function-blocking integrin-specific antibodies and celltransfection assays, Gavrilovskaya and co-workers showed that human allbb3 and avb3 integrins serve as receptors for pathogenic strains of hantavirus [37,38]. In contrast to pathogenic hantaviruses, nonpathogenic strains such as Prospect Hill virus (PHV) utilize b1 integrins. It has been shown that b3 integrins, rather than b1 integrins, have a significant role in vascular permeability; therefore, the use of b3 integrins by pathogenic hantaviruses could explain why infection by these strains results in severe disease. An additional point of interest is that pathogenic hantavirus– integrin association does not involve recognition of an RGD sequence nor does it require the presence of divalent metal cations, which indicates a distinct mode of receptor interaction. In confirmation of this, Raymond and colleagues provided strong evidence that the plexin–semaphorin– integrin domain (PSI), present at the apex of the bent/
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Figure 2. Ligand-binding sites on integrins. (a) The PSI domain (red) of the b3 subunit in the context of the bent avb3 integrin ectodomain structure. Pathogenic hantaviruses interact with the PSI domain of b3 integrins. (b) The avb3 domains involved in RGD binding are the a subunit b-propeller domain (dark blue) and the b subunit I domain (red). The crystal structure of avb3 complexed with an RGD peptide [64] reveals that the RGD binding site is between these two domains. Viruses that utilize the integrin RGD-binding site include adenovirus and the picornaviruses CAV9, echovirus 9, human parechoviruses and FMDV. (c) Composite integrin heterodimer structure depicting the ectodomain of an integrin containing an I domain in the a subunit modeled as in Figure 1c. The a I domain (dark blue), when it is present, contains the major ligand-binding site. EV1 interacts with the a2 I domain. In (a–c) the non-ligand-binding domains of the a subunit are shown in pink and those of the b subunit are shown in light blue, and the presumed location of the membrane is represented by a gray bar.
inactive form of the human b3 integrin subunit (Figure 2a), serves as the site for hantavirus binding [39]. The PSI domain is well removed from the site of RGD ligand binding, which is between the a subunit b-propeller domain and the b subunit I domain (Figure 2b). Thus the pathogenic hantaviruses have evolved a distinct strategy for interacting with integrins that, as far as we know, is not utilized by cellular proteins but nevertheless may provide additional clues to integrin signaling mechanisms. In this regard, substitution of a limited number of nonconserved amino acid residues in the homologous murine b3 integrin PSI domain, which does not bind hantavirus, for the corresponding residues in human b3, abrogated viral infection. The ability of hantavirus to bind to the b3 integrin in its inactive conformation on the surface of endothelial cells might prevent conversion of the integrin to its active state, thus explaining why this pathogen can alter vascular permeability. In support of this contention, it is well known that certain acute hemorrhagic diseases can arise from mutations in the b3 integrin subunit [40]. Members of the Reoviridae family utilize b3 and b1 integrins Among the members of the Reoviridae family of viruses, rotaviruses are large icosahedral, nonenveloped viruses containing a dsRNA genome. Rotaviruses are a major worldwide cause of infantile gastroenteritis that is associated with substantial morbidity and mortality. In a similar way to adenovirus cell entry, rotavirus entry into host cells occurs via a multistep process. An extended spike protein, known as VP4, mediates attachment of virus to cells via association with sialic acid residues [41]. Subsequent or simultaneous association of the VP4 with integrins a2b1, a4b1 or a4b7 promotes virus internalization [42–44]. An Asp, Gly, Glu (DGE) sequence in rotavirus VP4 mediates association of virus with integrin a2b1, as established by competition binding studies with recombinant protein/peptides. It should also be noted that the I domain of a2b1 is the site of integrin binding for rotavirus, a situation that also exists with certain echoviruses as discussed below. By contrast, the VP7 subunit appears to associate with www.sciencedirect.com
integrin axb2 as well as integrin avb3 to promote cell entry. Another member of the Reoviridae family, mammalian reovirus type 1 (Lang), has been shown to use b1 integrins for cell entry and infection. The spike protein of this virus, known as s1, associates with junctional adhesion molecule-1 (JAM-1), which enables high-affinity binding to cells [45]. Interestingly, expression of recombinant JAM-1 lacking the cytoplasmic tail failed to alter reovirus infection, which suggests that this virus uses secondary receptors for cell entry [46]. Because the l2 protein of reovirus contains both RGD and KGE integrin binding motifs, Dermody and colleagues [46] examined the potential role of these receptors in reovirus cell entry and infection. They found that b1 integrins selectively promote infection of mature reovirus, but not partially disassembled virions (ISVPs), and also facilitate virus internalization. However, the degree to which reovirus pathogenesis depends on association with integrin remains to be determined. Nonetheless, these findings underscore the similar modes of integrin usage by complex nonenveloped viruses, such as reovirus and adenovirus. Echovirus-1 interaction with the a2 I domain Several members of the Picornaviridae family, including echovirus-1 (EV1), use integrins as receptors during cell entry. EV1 is associated with a variety of human diseases, including meningitis, encephalitis, rash, respiratory infections and diarrhea. EV1 utilizes the a2b1 integrin [47], which is also one of the integrins that promotes rotavirus cell entry. The a2 integrin subunit contains an I domain, which when present usually serves as the major ligandbinding site (Figure 2c). Indeed it has been shown that the I domain is the crucial interaction domain for EV1 and that soluble receptor fragments containing the I domain are capable of preventing viral infection [48]. The I domain is also the interaction domain for a2b1 integrin with its physiological ligand collagen. However, the residues implicated in EV1 binding are mostly positioned on a different face of the a2 I domain from the collagen-binding site [49,50]. Another notable difference is that EV1 does not appear to require divalent cations for interaction with
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Figure 3. Model of interaction of EV1with integrin a2 I domain. (a) The EV1 capsid structure around the icosahedral fivefold axis (PDB ID 1EV1) [53] together with five copies of the a2 I domain (magenta, PDB ID 1AOX) [54] positioned approximately as modeled in the cryoEM study of the EV1–a2I complex [52]. The position of the rest of the integrin heterodimer is indicated by a black bar. (b) Side view of the modeled complex showing an a2 I domain fitting into a canyon on the surface of EV1.
a2b1, as does collagen, suggesting distinct interaction mechanisms for the association of viral and host ligands [51]. The structure of the complex of the a2 I domain with EV1 has been obtained by cryoEM [52]. The moderate ˚ ) cryoEM structure was interpreted with resolution (25 A the aid of the crystal structure for native EV1 (PDB ID 1EV1) [53] and the unliganded a2 I crystal structure (PDB ID 1AOX) [54]. Docking of the two crystal structures into the cryoEM density indicates that the I domain binds within the canyon on the surface of EV1. Both the N- and C-termini are oriented away from the virion surface, as expected, because in the full-length a2 integrin subunit the I domain is inserted within a loop of the sevenbladed b-propeller domain. Several favorable electrostatic interactions are predicted between a2I and surface residues of EV1. These interactions correlate well with EV1 residues that have been implicated in binding by studies with chimeric and mutated aI domains [49,55]. The MIDAS site of the I domain, which is normally involved in host–ligand interactions and interaction with collagen [7,8], does not appear to be in direct contact with the viral surface. The cryoEM-based model of the EV1–a2I complex
also suggests that the I domain cannot bind both collagen and EV1 simultaneously. Notably, the affinity of EV1 for the a2 I domain is about ten times greater than that of collagen type I. A homology model was built for approximately half of the a2b1 ectodomain and aligned with the EM-based model of the I domain bound to EV1 (Figure 3). This modeling study indicates that five integrins could potentially bind to one vertex region of the virus. Binding of EV1 to a2b1 initiates the subsequent internalization of the virus into caveolae [56]. Because integrin clustering is thought to have a role in triggering signal pathways inside the cell, Xing et al. [52] utilized confocal fluorescence microscopy to investigate whether EV1 caused integrin clustering on human osteosarcoma cells stably transfected with a2 integrin. Significant EV1-induced clustering was observed, which suggested that virus association with this receptor could lead to enhanced uptake as well as initiate cell signaling events. It should be noted that several other picornaviruses utilize integrins as receptors and these include coxsackievirus A9 (CAV9), echovirus 9 and 22, human parechoviruses and foot-and-mouth disease virus (FMDV) [47,57,58].
Figure 4. Comparison of RGD spacing in three viruses. (a) The vertex region of adenovirus 35F including the adenovirus 5 penton base (height color coded in red, orange and yellow) and adenovirus 35 fiber shaft (green). The RGD sites are located at the tops of protrusions (red) that project outward from the penton base. The density is from the 6.9 A˚ resolution cryoEM structure [65]. (b) The vertex region of FMDV with the disordered RGD loop region represented by dots. Adapted with permission from Ref. [66]. (c) The vertex region of CAV9 with the last two residues before the disordered RGD region represented by magenta spheres (PDB ID 1D4M) [67]. The spacing between the RGD sites around an icosahedral fivefold axis is 60 A˚ for all three viruses. (a,b) Modified with permission from Ref. [59]. www.sciencedirect.com
Review
TRENDS in Microbiology
However, unlike EV1, these other picornaviruses interact with av integrins, which do not contain an I domain, and they interact via conventional integrin-binding RGD motifs. A striking feature of nonenveloped virus interactions with integrins is that this often involves very similar geometry/spacing of receptor engagement around the fivefold axis of the virion. The correspondence of the spacing ˚ ) of the five RGD loops around a single vertex of (60 A adenovirus with that of FMDV has been noted [59]. This is despite the fact that adenovirus and FMDV are structurally and evolutionarily distinct, and are from two different virus families, Adenoviridae and Picornaviridae. Less surprisingly FMDV and CAV9, both members of the picornavirus family, have a similar spacing for their integrinbinding RGD sites. The RGD sites of all three viruses ˚ and (adenovirus, FMDV and CAV9) are spaced by 60 A are found on mobile loops or peptide regions (Figure 4). Presumably this spacing is optimal for interaction of the virus with multiple integrins at a single capsid vertex. Perhaps the close packing of integrins induces a larger conformational change that is transmitted to the cytoplasmic integrin tails than would be induced by isolated RGD–integrin interactions. Conclusions and future perspectives It is not surprising that numerous viruses (as well as bacteria) have usurped integrins for cell invasion, because integrins are expressed on a wide variety of cells throughout the body. Moreover, integrin ligation by microbial pathogens elicits potent signaling responses that promote cytoskeletal reorganization and/or cell entry. Despite the obvious advantages of using integrins as entry receptors, viruses also use non-integrin receptors, such as members of the Ig superfamily (e.g. CAR and JAM-1). It is important to note that these receptors allow high-affinity binding of virions to cells but do not trigger cell signaling or promote entry. The different choices viruses make with respect to receptor usage may reflect the ‘trial and error’ nature of evolution or perhaps different requirements for virus replication in certain types of cells displaying integrins or non-integrin receptors. There are several key questions regarding virus–integrin interactions that have yet to be answered (Box 2). Both enveloped and nonenveloped viruses utilize integrins for cell invasion and they do so with a variety of mechanisms. One interesting observation that has emerged from recent investigations is that integrin avb3, in particular, which is known to exhibit substantial
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Box 2. Outstanding questions Does the precise spatial orientation of integrin-binding motifs on virus particles regulate cell signaling and infection/entry? What are the key signaling molecules that are common in the cellentry pathways of diverse viruses? Why are certain integrins used by a particular virus but not by others? Why is avb3 used by multiple virus families? Do integrins need to be in ‘active’ conformation for virus association and/or cell signaling? Are there novel integrin-binding sequences on viral proteins that remain to be discovered? Can integrin antagonists be made clinically useful for the treatment of viral infections?
diversity in ligand recognition [1], is one of the most comonly used receptors by members of diverse virus families (Table 1). It is, therefore, tempting to speculate that multiple viruses have evolved to take advantage of the promiscuous attributes of integrin avb3. Because integrins such as avb3 are capable of recognizing relatively small peptide sequences, this may provide a convenient pathway for virus evolution as integrin ligand sequences could be readily incorporated without drastically altering virus structure. Clearly the long co-evolution of viruses with their hosts has led to the variations on a theme that we are now discovering about virus–integrin interactions. Although much is understood now about integrin structure, questions still remain about the precise conformational changes that integrins must undergo to achieve both inside–out and outside–in signaling. The rapid progress made in recent years continues to aid our understanding of integrin structure. We anticipate that additional structural studies of virus–integrin complexes will help to elucidate the nature of the conformational changes induced by multivalent viral ligands and the diverse molecular mechanisms that can be involved in integrin signaling. Integrins also remain attractive drug targets for interfering with cell proliferation, migration or tissue localization of inflammatory, angiogenic and tumor cells [60,61]. Integrin-targeted drugs [62] might also modulate virus– ligand affinity and signaling, a situation that could prove useful in controlling infectious diseases. We anticipate that further investigation of virus–integrin interactions will contribute not only to a broader understanding of viral pathogenesis but also of integrin physiology. Such knowledge could eventually lead to the development of new antiviral as well as integrin-related therapeutic compounds.
Table 1. Integrins used by different viruses Virus family Adenovirus Herpesvirus Hantavirus Picornavirus
Reoviridae
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Virus type Adenovirus 2/5 HCMV HHV-8 SNV PHV EV1 FMDV CA9 EV9 Rotavirus Reovirus (Lang)
Integrins used avb1, avb3, aMb2, avb5 b1, avb3 a3b1, a2b1 allbb3, avb3 b1 a2b1 a5b1, avb3 avb3, avb6 avb3 axb2, a2b1, avb1, a4b7, avb3 b1
Role in infection Cell entry, signaling, endosome escape Cell entry, signaling Cell entry, signaling Attachment, entry (?) Attachment, entry (?) Attachment, signaling, entry Attachment, entry Attachment, entry Attachment, entry Attachment, entry Cell entry
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References 1 Hynes, R.O. (1992) Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25 2 Cheresh, D.A. and Spiro, R.C. (1987) Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen and von Willebrand factor. J. Biol. Chem. 262, 17703–17711 3 Fox, G. et al. (1989) The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). J. Gen. Virol. 70, 625–637 4 Wickham, T.J. et al. (1993) Integrins avb3 and avb5 promote adenovirus internalization but not virus attachment. Cell 73, 309–319 5 Vogel, B.E. et al. (1990) A novel fibronectin receptor with an unexpected subunit composition (avb1). J. Biol. Chem. 265, 5934– 5937 6 Roivainen, M. et al. (1991) RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases. J. Virol. 65, 4735–4740 7 Luo, B.H. et al. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619–647 8 Arnaout, M.A. et al. (2005) Integrin structure, allostery, and bidirectional signaling. Annu. Rev. Cell Dev. Biol. 21, 381–410 9 Xiong, J.P. et al. (2001) Crystal structure of the extracellular segment of integrin avb3. Science 294, 339–345 10 Xiong, J-P. et al. (2004) A novel adaptation of the integrin PSI domain revealed from its crystal structure. J. Biol. Chem. 279, 40252–40254 11 Suzuki, S. and Naitoh, Y. (1990) Amino acid sequence of a novel integrin b4 subunit and primary expression of the mRNA in epithelial cells. EMBO J. 9, 757–763 12 Vinogradova, O. et al. (2002) A structural mechanism of integrin alphaIIb beta 3 ‘‘Inside-Out’’ activation as regulated by its cytoplasmic face. Cell 110, 587–597 13 Wegener, K.L. et al. (2007) Structural basis of integrin activation by talin. Cell 128, 171–182 14 Wickham, T.J. et al. (1994) Integrin avb5 selectively promotes adenovirus mediated cell membrane permeabilization. J. Cell Biol. 127, 257–264 15 Bai, M. et al. (1994) Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2. J. Virol. 68, 5925–5932 16 Mathias, P. et al. (1994) Multiple adenovirus serotypes use av integrins for infection. J. Virol. 68, 6811–6814 17 Li, E. et al. (2001) Integrin avß1 is an adenovirus co-receptor. J. Virol. 75, 5405–5409 18 Davison, E. et al. (1997) Integrin a5b1-mediated adenovirus infection is enhanced by the integrin-activating antibody TS2/16. J. Virol. 71, 6204–6207 19 Chiu, C.Y. et al. (1999) Structure of adenovirus complexed with its internalization receptor, avb5 integrin. J. Virol. 73, 6759–6768 20 Stewart, P.L. et al. (1997) Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization. EMBO J. 16, 1189–1198 21 Kari, B. and Gehrz, R. (1992) A human cytomegalovirus glycoprotein complex designated gC-II is a major heparin-binding component of the envelope. J. Virol. 66, 1761–1764 22 Compton, T. et al. (1993) Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193, 834–841 23 Spear, P.G. and Longnecker, R. (2003) Herpesvirus entry: an update. J. Virol. 77, 10179–10185 24 Kowalik, T.F. et al. (1993) Multiple mechanisms are implicated in the regulation of NF-kappa B activity during human cytomegalovirus infection. Proc. Natl Acad. Sci. USA 90, 1107–1111 25 Feire, A.L. et al. (2004) Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc. Natl Acad. Sci. USA 101, 15470–15475 26 Mochizuki, S. and Okada, Y. (2007) ADAMs in cancer cell proliferation and progression. Cancer Sci. 98, 621–628 27 Wang, X. et al. (2005) Integrin avb3 is a coreceptor for human cytomegalovirus. Nat. Med. 11, 515–521 28 Parry, C. et al. (2005) Herpes simplex virus type 1 glycoprotein H binds to alphavbeta3 integrins. J. Gen. Virol. 86, 7–10 www.sciencedirect.com
29 Akula, S.M. et al. (2001) Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties. Virology 284, 235–249 30 Akula, S.M. et al. (2003) Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis. J. Virol. 77, 7978–7990 31 Akula, S.M. et al. (2002) Integrin a3b1 (CD 49c/29) is a cellular receptor for Kaposi’s Sarcoma-Associated Herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108, 407–419 32 Veettil, M.V. et al. (2006) RhoA-GTPase facilitates entry of Kaposi’s sarcoma-associated herpesvirus into adherent target cells in a Srcdependent manner. J. Virol. 80, 11432–11446 33 Krishnan, H.H. et al. (2006) Focal adhesion kinase is critical for entry of Kaposi’s Sarcoma-associated herpesvirus into target cells. J. Virol. 80, 1167–1180 34 Parks, W.C. (2007) What is the alpha2beta1 integrin doing in the epidermis? J. Invest. Dermatol. 127, 264–266 35 Kaleeba, J.A.R. and Berger, E.A. (2006) Kaposi’s sarcoma-associated herpesvirus fusion-entry receptor: Cystine transporter xCT. Science 311, 1921–1924 36 Feral, C.C. et al. (2005) CD98hc (SLC3A2) mediates integrin signaling. Proc. Natl Acad. Sci. USA 102, 355–360 37 Gavrilovskaya, I.N. et al. (1998) b3 integrins mediate the cellular entry of hantaviruses that cause respiratory failure. Proc. Natl Acad. Sci. USA 95, 7074–7079 38 Gavrilovskaya, I.N. et al. (1999) Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by b3 integrins. J. Virol. 73, 3951–3959 39 Raymond, T. et al. (2005) Pathogenic hantaviruses bind plexinsemaphorin-integrin domains present at the apex of inactive, bent avb3 integrin conformers. Proc. Natl Acad. Sci. USA 102, 1163–1168 40 Hodivala-Dilke, K.M. et al. (1999) b3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Invest. 103, 229–238 41 Pesavento, J.B. et al. (2005) pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization. J. Virol. 79, 8572–8580 42 Lo´pez, S. and Arias, C.F. (2004) Multistep entry of rotavirus into cells: a Versaillesque dance. Trends Microbiol. 12, 271–278 43 Graham, K.L. et al. (2006) Rotavirus spike protein VP5* binds alpha2beta1 integrin on the cell surface and competes with virus for cell binding and infectivity. J. Gen. Virol. 87, 1275–1283 44 Graham, K.L. et al. (2005) Rotaviruses interact with alpha4beta7 and alpha4beta1 integrins by binding the same integrin domains as natural ligands. J. Gen. Virol. 86, 3397–3408 45 Guglielmi, K.M. et al. (2006) Attachment and cell entry of mammalian orthoreovirus. Curr. Top. Microbiol. Immunol. 309, 1–38 46 Maginnis, M.S. et al. (2006) Beta1 integrin mediates internalization of mammalian reovirus. J. Virol. 80, 2760–2770 47 Evans, D.J. and Almond, J.W. (1998) Cell receptors for picornaviruses as determinants of cell tropism and pathogenesis. Trends Microbiol. 6, 198–202 48 King, S.L. et al. (1995) Echovirus 1 interaction with the isolated VLA-1 I domain. J. Virol. 69, 3237–3239 49 King, S.L. et al. (1997) Echovirus 1 interaction with the human very late antigen-2 (integrin alpha2beta1) I domain. Identification of two independent virus contact sites distinct from the metal ion-dependent adhesion site. J. Biol. Chem. 272, 28518–28522 50 Kamata, T. et al. (1994) Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29). J. Biol. Chem. 269, 9659–9663 51 Bergelson, J.M. et al. (1993) The integrin VLA-2 binds echovirus 1 and extracellular matrix ligands by different mechanisms. J. Clin. Invest. 92, 232–239 52 Xing, L. et al. (2004) Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1. J. Biol. Chem. 279, 11632–11638 53 Filman, D.J. et al. (1998) Structure determination of echovirus 1. Acta Crystallogr. D Biol. Crystallogr. 54, 1261–1272 54 Emsley, J. et al. (1997) Crystal structure of the I domain from integrin a 2b1. J. Biol. Chem. 272, 28512–28517 55 Dickeson, S.K. et al. (1999) Determinants of ligand binding specificity of the a1b1 and a2b2 integrin. J. Biol. Chem. 274, 32182–32191
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56 Pietia¨inen, V. et al. (2004) Echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin II, and signaling events. Mol. Biol. Cell 15, 4911–4925 57 Whitton, J.L. et al. (2005) Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Immunol. 3, 765–776 58 Nelsen-Salz, B. et al. (1999) Integrin avb3 (vitronectin receptor) is a candidate receptor for the virulent echovirus 9 strain Barty. J. Gen. Virol. 80, 2311–2313 59 Nemerow, G.R. and Stewart, P.L. (2001) Antibody neutralization epitopes and integrin binding sites on nonenveloped viruses. Virology 288, 189–191 60 Staunton, D.E. et al. (2006) Targeting integrin structure and function in disease. Adv. Immunol. 91, 111–157 61 Lu, X. et al. (2006) Integrins in drug targeting-RGD templates in toxins. Curr. Pharm. Des. 12, 2749–2769 62 Yin, H. et al. (2007) Computational design of peptides that target transmembrane helices. Science 315, 1817–1822 63 Adair, B.D. et al. (2005) Three-dimensional EM structure of the ectodomain of integrin av b3 in a complex with fibronectin. J. Cell Biol. 168, 1109–1118
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64 Xiong, J-P. et al. (2002) Crystal structure of the extracellular segment of integrin avß3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 65 Saban, S.D. et al. (2006) Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments. J. Virol. 80, 12049– 12059 66 Acharya, R. et al. (1989) The three-dimensional structure of foot-and˚ resolution. Nature 337, 709–716 mouth disease virus at 2.9 A 67 Hendry, E. et al. (1999) The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses. Structure 7, 1527–1538 68 Li, E. et al. (1998) Adenovirus endocytosis via av integrins requires phosphoinositide-3-OH-kinase. J. Virol. 72, 2055–2061 69 Philpott, N.J. et al. (2004) Adenovirus-induced maturation of dendritic cells through a P13 kinase-mediated TNF-a induction pathway. Proc. Natl. Acad. Sci. U. S. A. 101, 6200–6205 70 Rajala, M.S. et al. (2005) Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation. J. Virol. 79, 12332–12341
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Cell integrins: commonly used receptors for diverse viral pathogens Phoebe L. Stewart1 and Glen R. Nemerow2 1 2
Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
When searching for their favorite host tissues, animal viruses frequently attach to cell-surface receptors that have key roles in normal cell physiology. Integrins are prime examples of physiologically important receptors that have been usurped by nonenveloped and enveloped viruses for attachment and/or cell entry. This family of heterodimeric receptors mediates cell adhesion, cell migration, tumor metastasis and cell differentiation. Recent investigations have shed new light on integrin structure as well as on the underlying molecular features of their association with viral ligands. In this review, we discuss several examples of virus–integrin interactions that highlight recent advances in this field. The continuing improvements in virus and cell imaging techniques have helped to uncover the molecular basis of how integrins are recognized by such a wide range of microbial pathogens to invade host cells. Integrin structure Integrins are integral membrane proteins, and all a and b subunits contain a single transmembrane spanning helix [1]. Many, but not all, integrins recognize RGD sequences displayed on the exposed loops of viral capsid proteins or extracellular matrix (ECM) proteins [2–6]. Virus or ECM binding induces clustering and/or conformational changes in integrin quaternary structure (for excellent recent reviews, see Refs [7,8]). Conformational changes to integrin can elicit cell-signaling events that increase ligand affinity/avidity as well as promote cytoskeletal rearrangement and virus internalization. As membrane proteins, integrins present significant challenges for structural biology. In addition, integrins undergo conformational changes to achieve outside–in and inside–out signaling, which adds an additional layer of complexity. Although there is still controversy, particularly regarding the extent of conformational change [7,8], structural information has been obtained by a variety of techniques, including X-ray crystallography, NMR, electron microscopy (EM) and three-dimensional image reconstruction of negatively stained and cryopreserved samples. The extracellular portion, or ectodomain, of a and b subunits typically contains 700–1000 amino acids. Alpha subunits contain four or five extracellular domains and a small cytoplasmic domain (Figure 1a). Half of the 18 different a subunits (aD, aE, aL, aM, aX, a1, a2, a10, a11) contain an I domain, also Corresponding author: Nemerow, G.R. ([email protected]). Available online 7 November 2007. www.sciencedirect.com
referred to as a von Willebrand factor A domain, or simply an A domain. When an I domain is present in an a subunit, it is nearly always the ligand-binding site. When the I domain is absent from the a subunit (as in a3, a4, a5, a6, a7, a8, a9, av and aIIb), then the ligand-binding site is typically formed by the b-propeller domain of the a subunit together with an I domain of the b subunit. Beta subunits (b1–b8) contain eight extracellular domains, including an I domain, and a cytoplasmic domain of various lengths (Figure 1b). There are several crystal structures of the a subunit I domain both with and without bound ligands, such as a triple-helical collagen peptide and fragments of intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 [7]. The a I domain has a conserved metal-ion-dependent adhesion site (MIDAS) that binds Mg2+ under physiological conditions, and the residues that coordinate the metal and surround the metal-binding site are important for ligand binding. The conformational changes of the a I domain that occur upon ligand binding involve the shift of an a-helix (a7) away from the ligand-binding site [7]. The b subunit I domain has a similar structure with three metal binding sites: MIDAS, adjacent MIDAS (ADMIDAS) and ligandinduced metal-binding site (LIMBS) [7]. The crystal structure of the extracellular portion, or ectodomain, of avb3 provides a detailed picture of an integrin without an a I domain and reveals a bent conformation for both the a and b subunits [9,10] (Figure 1c). Some studies suggest that a large conformational change, involving straightening of the integrin ectodomain, accompanies RGD ligand binding (the ‘switchblade’ model), whereas other studies suggest a more subtle conformational change without extension (the ‘deadbolt’ model) [7,8]. There is a consensus, however, that some conformational change must occur upon ligand binding to convey a signal from ‘outside–in’. The cytoplasmic domains of the a and b subunits interact with a variety of intracellular proteins, such as kinases and cytoskeletal proteins to promote signaling. The cytoplasmic domains of a and b subunits are all relatively short (20–50 amino acids) with the exception of b4, which has a long cytoplasmic domain of 1000 amino acids and contains four fibronectin type III repeat domains [11]. NMR studies have provided structures of several a subunit and b subunit cytoplasmic tails, as well as the complex of full-length aIIb and b3 cytoplasmic tails [12]. The complex structure shows multiple hydrophobic and electrostatic contacts within the membrane-proximal
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Figure 1. Integrin domains and heterodimer structure. (a) The structural domains of a subunits with four or five extracellular domains, a transmembrane spanning region and a small cytoplasmic domain. The I domain (magenta) is present only in nine of the 18 different a subunits. The N-terminus is near the b-propeller domain and the C-terminus is cytoplasmic. (b) The structural domains of b subunits with eight extracellular domains, a transmembrane spanning region and cytoplasmic domain. The EGF-1 and EGF-2 domains (gray) are models based on the EGF-3 domain structure in the avb3 crystal structure [10]. The N-terminus is near the PSI domain and the C-terminus is cytoplasmic. The extended domain representations of the a and b subunits shown in (a) and (b) are based on figures in Refs [7,8]. (c) Composite integrin heterodimer structure (bent conformation) based on the crystal structure of the a2 I domain (PDB ID 1AOX) [54], the crystal structure of the avb3 ectodomain (PDB ID 1U8C) [10] and the NMR structure of the cytoplasmic tails of aIIbb3 (PDB ID 1M8O) [12]. The a subunit is shown as mostly blue, with an associated I domain in magenta. The b subunit is shown as red. The EGF-1 and EGF-2 domains, which are missing from the avb3 structure, are not shown. The position of the a I domain is approximate and its effect on the overall fold of the ectodomain is assumed to be minimal for this composite structural model. The orientation of the extracellular domains relative to the membrane is as modeled in an EM study of the avb3 ectodomain complexed with four domains of fibronectin [63]. The membrane is represented by a gray bar, 30 A˚ thick on the molecular scale. The domains are shown as 10 A˚ resolution filtered density maps based on their atomic coordinates.
helices of the a and b cytoplasmic tails. A recent structural study has shed new light on the structural basis of ‘inside– out’ signaling. The intracellular protein talin, which binds to b1, b2 and b3 integrins, connects integrins to the actin cytoskeleton and can alter the affinity of integrins for extracellular ligands. The NMR structure of a talin–integrin complex, combined with analysis of targeted mutations, reveals that the contact between a subdomain of talin and the membrane-proximal region of the b subunit tail is crucial for activation of integrin [13]. Interaction of adenovirus with av integrins Adenovirus was one of the first examples of a virus that was shown to use multiple cell receptors to infect host cells. After attachment of adenovirus to cells via association with coxsackie adenovirus receptor (CAR), various adenovirus serotypes utilize av integrins and b1 integrins for cell entry [4,14–18]. Adenoviruses interact with av integrins via a long flexible RGD loop on the surface of the penton base. The structures of adenovirus types 2 and 12 in complex with a soluble form of avb5 integrin have been determined ˚ ) resolution by cryoelectron microscopy to moderate (21 A (cryoEM) [19]. These structures reveal a ring of integrin density over the five RGD loops of the penton base. Although these cryoEM structures have imposed icosahedral symmetry and represent the average of datasets with 200–500 particle images, they do suggest that multiple integrins can bind at each penton base. This concept was supported by a kinetic analysis (BIAcore) of interactions of adenovirus 2 with avb5 that indicated that four or five integrin molecules bound per penton base at close to saturation. Adenovirus is capable of escaping neutralization by antibodies directed to the RGD sites [20]. This is probably attributable to the shielding effect of the www.sciencedirect.com
adenovirus fiber, which protrudes from the penton base. These structural analyses provided insight into the role of integrin clustering in adenovirus entry. In particular, they led to studies of the relationship between integrin signaling and virus entry and infection (Box 1). Herpesviruses exploit integrins for cell entry and infection In a somewhat surprising development, several large enveloped DNA viruses have been shown to use integrins to invade host cells. Human cytomegalovirus (HCMV), a b-herpesvirus and the most frequent cause of congenital birth defects, initially binds to host cells via association with cell-surface heparan sulfate proteoglycans [21,22]. However, this event is not sufficient to allow HCMV entry, and thus further investigations have sought to identify other receptors that mediate cell entry. Among the many outer envelope glycoproteins of human herpesviruses, one of these, designated gB, is required for subsequent cell entry/fusion [23]. Because HCMV infection is associated with cell signaling events [24] characteristic of integrin ligation, Feire and colleagues [25] searched the sequence of HCMV gB for conventional integrin-binding motifs (i.e. RGD, LDV, DGE) but did not find these sequences. Instead, they noted that gB contains the RX6–8DLXXF consensus sequence present in the ADAM (a disintegrin and a metalloprotease) family of proteins, which recognize integrins, particularly b1 subtypes [26]. This so-called disintegrin sequence is preserved in the gB glycoproteins of many clinical and laboratory strains of HCMV as well as in other g- and b-herpesviruses but not in a- herpesviruses. Using integrin-function-blocking antibodies, peptide antagonists and integrin-deficient cell lines, Feire and co-workers demonstrated that several b1 as well as
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Box 1. Integrins as transducers of cell signaling during virus entry Integrins are heterodimeric cell-surface glycoproteins comprising an a and b subunit. Their association with extracellular matrix proteins or viral capsid proteins can initiate multiple signaling events that facilitate receptor-mediated endocytosis of virus particles. The cytoplasmic domains of b1 and b3 integrin subunits, in particular, associate with several host-cell signaling molecules upon binding to the RGD motifs displayed on the surface of viral ligands. Adenovirus provided one of the first examples of signaling events via ligation of integrin [68]. The association of the adenovirus penton base with av integrins induces receptor clustering, which leads to the tyrosine phosphorylation/activation of focal adhesion kinase (FAK). FAK is well known for its role in cell motility as well as for its impact on downstream molecules such as ERK1/ERK2 mitogen-activated protein kinases (MAP kinases). Interestingly, adenovirus signaling to FAK does not result in enhanced virus entry into cells. Instead, FAK activation might contribute to the production of proinflammatory cytokines [69], a situation that limits the use of adenovirus vectors for systemic delivery in humans. Adenovirus-mediated ligation of integrin also promotes activation of p130CAS, PI3K and the Rho family of small GTPases. One consequence of these signaling events is actin polymerization and enhanced adenovirus internalization. In addition to enhanced endocytosis, PI3K activation might result in increased survival of certain host cells through subsequent signaling to protein kinase B (AKT). This might allow completion of the virus replication cycle and production of progeny virions [70]. Thus, viruses such as adenovirus elicit potent signaling events via ligation of integrin that enhance infection of different host cells. These same signaling events can also affect host immune responses as well as cell survival and cell proliferation.
avb3 integrins engage HCMV gB through its disintegrin motif, thereby facilitating cell entry and infection [25]. Moreover, engagement of integrins by HCMV leads to phosphorylation of the cell-signaling molecule, focal adhesion kinase (FAK) as well as polymerization of the actin cytoskeleton, similar to other integrin-binding viruses, such as adenovirus and Kaposi’s sarcoma-associated herpesvirus (KSHV). Wang and colleagues have also provided evidence for the use of integrin avb3 by HCMV in an RGD-independent manner [27]. Interestingly, it is the gH envelope protein of HCMV, rather than the gB protein, that recognizes integrin avb3 in an RGD-dependent manner. This situation has also been reported to occur with herpes simplex virus type 1 (HSV-1) association with avb3 integrin [28]. Further analyses are needed to uncover the precise structural interactions of HMCV gB with its integrin partners as well as to determine how integrin engagement affects HCMV pathogenesis. Another member of the herpesvirus family, HHV-8, is frequently associated with Kaposi’s sarcoma in patients with HIV/AIDS and is referred to as KSHV. As is the case for most herpesviruses, KSHV uses heparan sulfate for initial cell adhesion [29], whereas entry into cells via endocytosis [30] requires interaction of KSHV gB with integrin a3b1 [31]. Interestingly, Akula and colleagues [31] suggested that a region of the KSHV containing an RGD sequence was important for integrin association on the basis of competition studies with RGD peptides and anti-RGD antibodies. Since these reagents only partially inhibited KSHV infection, it is possible that the disintegrin motif that is also present in KSHV gB [25] contributes to integrin associations. Regardless of the precise mode of www.sciencedirect.com
engagement of integrin, KSHV induces activation of the cell-signaling molecules, RhoA GTPase and Src [32] as well as FAK [33], that promote virus entry and infection. An intriguing question arising from these findings is whether KSHV/integrin-mediated cell signaling might have important consequences for viral pathogenesis. In particular, integrin-mediated cell signaling events could promote the secretion of growth factors and/or cytokines that facilitate epithelial cell proliferation, a situation that could accelerate Kaposi sarcoma tumor formation. In support of this possibility, ligation of integrin a2b1 by certain ECM proteins allows re-epithelization of human skin wounds, a process that requires cell migration and/or proliferation [34]. Thus, whether engagement of related b1 integrins, including a3b1, by KSHV has a significant role in the development of Kaposi sarcoma remains to be determined. Recently, Kaleeba and Berger reported that KSHV uses a distinct receptor for cell membrane fusion after integrinmediated virus uptake [35]. They demonstrated that a 501 amino acid membrane protein known as xCT (the 12transmembrane light chain of the human cysteine–glutamate exchange transporter system, Xc) promotes KSHV membrane fusion with host cells, an event required for successful virus infection. Thus, cells originally resistant to membrane fusion and infection by KSHV could be rendered susceptible by transient expression of xCT. Interestingly, members of the xCT family have been shown to be part of a heterodimeric complex including the 80 kDa CD98 (4F2 antigen) and a 45 kDa light chain including xCT that is capable of regulating b1 integrin signaling [36]. These studies illustrate the interplay between integrins and their associated partners in transmitting cell signals that promote distinct steps in viral invasion. b3 Integrins serve as receptors of pathogenic hantaviruses Hantaviruses are enveloped viruses containing a negative-stranded RNA genome. Several members of this virus family, such as Sin Nombre virus (SNV), cause two highly lethal diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS). Hantaviral infection is associated with disruption of endothelial and platelet functions, which result in clotting defects and acute pulmonary edema. Using competition assays with function-blocking integrin-specific antibodies and celltransfection assays, Gavrilovskaya and co-workers showed that human allbb3 and avb3 integrins serve as receptors for pathogenic strains of hantavirus [37,38]. In contrast to pathogenic hantaviruses, nonpathogenic strains such as Prospect Hill virus (PHV) utilize b1 integrins. It has been shown that b3 integrins, rather than b1 integrins, have a significant role in vascular permeability; therefore, the use of b3 integrins by pathogenic hantaviruses could explain why infection by these strains results in severe disease. An additional point of interest is that pathogenic hantavirus– integrin association does not involve recognition of an RGD sequence nor does it require the presence of divalent metal cations, which indicates a distinct mode of receptor interaction. In confirmation of this, Raymond and colleagues provided strong evidence that the plexin–semaphorin– integrin domain (PSI), present at the apex of the bent/
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Figure 2. Ligand-binding sites on integrins. (a) The PSI domain (red) of the b3 subunit in the context of the bent avb3 integrin ectodomain structure. Pathogenic hantaviruses interact with the PSI domain of b3 integrins. (b) The avb3 domains involved in RGD binding are the a subunit b-propeller domain (dark blue) and the b subunit I domain (red). The crystal structure of avb3 complexed with an RGD peptide [64] reveals that the RGD binding site is between these two domains. Viruses that utilize the integrin RGD-binding site include adenovirus and the picornaviruses CAV9, echovirus 9, human parechoviruses and FMDV. (c) Composite integrin heterodimer structure depicting the ectodomain of an integrin containing an I domain in the a subunit modeled as in Figure 1c. The a I domain (dark blue), when it is present, contains the major ligand-binding site. EV1 interacts with the a2 I domain. In (a–c) the non-ligand-binding domains of the a subunit are shown in pink and those of the b subunit are shown in light blue, and the presumed location of the membrane is represented by a gray bar.
inactive form of the human b3 integrin subunit (Figure 2a), serves as the site for hantavirus binding [39]. The PSI domain is well removed from the site of RGD ligand binding, which is between the a subunit b-propeller domain and the b subunit I domain (Figure 2b). Thus the pathogenic hantaviruses have evolved a distinct strategy for interacting with integrins that, as far as we know, is not utilized by cellular proteins but nevertheless may provide additional clues to integrin signaling mechanisms. In this regard, substitution of a limited number of nonconserved amino acid residues in the homologous murine b3 integrin PSI domain, which does not bind hantavirus, for the corresponding residues in human b3, abrogated viral infection. The ability of hantavirus to bind to the b3 integrin in its inactive conformation on the surface of endothelial cells might prevent conversion of the integrin to its active state, thus explaining why this pathogen can alter vascular permeability. In support of this contention, it is well known that certain acute hemorrhagic diseases can arise from mutations in the b3 integrin subunit [40]. Members of the Reoviridae family utilize b3 and b1 integrins Among the members of the Reoviridae family of viruses, rotaviruses are large icosahedral, nonenveloped viruses containing a dsRNA genome. Rotaviruses are a major worldwide cause of infantile gastroenteritis that is associated with substantial morbidity and mortality. In a similar way to adenovirus cell entry, rotavirus entry into host cells occurs via a multistep process. An extended spike protein, known as VP4, mediates attachment of virus to cells via association with sialic acid residues [41]. Subsequent or simultaneous association of the VP4 with integrins a2b1, a4b1 or a4b7 promotes virus internalization [42–44]. An Asp, Gly, Glu (DGE) sequence in rotavirus VP4 mediates association of virus with integrin a2b1, as established by competition binding studies with recombinant protein/peptides. It should also be noted that the I domain of a2b1 is the site of integrin binding for rotavirus, a situation that also exists with certain echoviruses as discussed below. By contrast, the VP7 subunit appears to associate with www.sciencedirect.com
integrin axb2 as well as integrin avb3 to promote cell entry. Another member of the Reoviridae family, mammalian reovirus type 1 (Lang), has been shown to use b1 integrins for cell entry and infection. The spike protein of this virus, known as s1, associates with junctional adhesion molecule-1 (JAM-1), which enables high-affinity binding to cells [45]. Interestingly, expression of recombinant JAM-1 lacking the cytoplasmic tail failed to alter reovirus infection, which suggests that this virus uses secondary receptors for cell entry [46]. Because the l2 protein of reovirus contains both RGD and KGE integrin binding motifs, Dermody and colleagues [46] examined the potential role of these receptors in reovirus cell entry and infection. They found that b1 integrins selectively promote infection of mature reovirus, but not partially disassembled virions (ISVPs), and also facilitate virus internalization. However, the degree to which reovirus pathogenesis depends on association with integrin remains to be determined. Nonetheless, these findings underscore the similar modes of integrin usage by complex nonenveloped viruses, such as reovirus and adenovirus. Echovirus-1 interaction with the a2 I domain Several members of the Picornaviridae family, including echovirus-1 (EV1), use integrins as receptors during cell entry. EV1 is associated with a variety of human diseases, including meningitis, encephalitis, rash, respiratory infections and diarrhea. EV1 utilizes the a2b1 integrin [47], which is also one of the integrins that promotes rotavirus cell entry. The a2 integrin subunit contains an I domain, which when present usually serves as the major ligandbinding site (Figure 2c). Indeed it has been shown that the I domain is the crucial interaction domain for EV1 and that soluble receptor fragments containing the I domain are capable of preventing viral infection [48]. The I domain is also the interaction domain for a2b1 integrin with its physiological ligand collagen. However, the residues implicated in EV1 binding are mostly positioned on a different face of the a2 I domain from the collagen-binding site [49,50]. Another notable difference is that EV1 does not appear to require divalent cations for interaction with
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Figure 3. Model of interaction of EV1with integrin a2 I domain. (a) The EV1 capsid structure around the icosahedral fivefold axis (PDB ID 1EV1) [53] together with five copies of the a2 I domain (magenta, PDB ID 1AOX) [54] positioned approximately as modeled in the cryoEM study of the EV1–a2I complex [52]. The position of the rest of the integrin heterodimer is indicated by a black bar. (b) Side view of the modeled complex showing an a2 I domain fitting into a canyon on the surface of EV1.
a2b1, as does collagen, suggesting distinct interaction mechanisms for the association of viral and host ligands [51]. The structure of the complex of the a2 I domain with EV1 has been obtained by cryoEM [52]. The moderate ˚ ) cryoEM structure was interpreted with resolution (25 A the aid of the crystal structure for native EV1 (PDB ID 1EV1) [53] and the unliganded a2 I crystal structure (PDB ID 1AOX) [54]. Docking of the two crystal structures into the cryoEM density indicates that the I domain binds within the canyon on the surface of EV1. Both the N- and C-termini are oriented away from the virion surface, as expected, because in the full-length a2 integrin subunit the I domain is inserted within a loop of the sevenbladed b-propeller domain. Several favorable electrostatic interactions are predicted between a2I and surface residues of EV1. These interactions correlate well with EV1 residues that have been implicated in binding by studies with chimeric and mutated aI domains [49,55]. The MIDAS site of the I domain, which is normally involved in host–ligand interactions and interaction with collagen [7,8], does not appear to be in direct contact with the viral surface. The cryoEM-based model of the EV1–a2I complex
also suggests that the I domain cannot bind both collagen and EV1 simultaneously. Notably, the affinity of EV1 for the a2 I domain is about ten times greater than that of collagen type I. A homology model was built for approximately half of the a2b1 ectodomain and aligned with the EM-based model of the I domain bound to EV1 (Figure 3). This modeling study indicates that five integrins could potentially bind to one vertex region of the virus. Binding of EV1 to a2b1 initiates the subsequent internalization of the virus into caveolae [56]. Because integrin clustering is thought to have a role in triggering signal pathways inside the cell, Xing et al. [52] utilized confocal fluorescence microscopy to investigate whether EV1 caused integrin clustering on human osteosarcoma cells stably transfected with a2 integrin. Significant EV1-induced clustering was observed, which suggested that virus association with this receptor could lead to enhanced uptake as well as initiate cell signaling events. It should be noted that several other picornaviruses utilize integrins as receptors and these include coxsackievirus A9 (CAV9), echovirus 9 and 22, human parechoviruses and foot-and-mouth disease virus (FMDV) [47,57,58].
Figure 4. Comparison of RGD spacing in three viruses. (a) The vertex region of adenovirus 35F including the adenovirus 5 penton base (height color coded in red, orange and yellow) and adenovirus 35 fiber shaft (green). The RGD sites are located at the tops of protrusions (red) that project outward from the penton base. The density is from the 6.9 A˚ resolution cryoEM structure [65]. (b) The vertex region of FMDV with the disordered RGD loop region represented by dots. Adapted with permission from Ref. [66]. (c) The vertex region of CAV9 with the last two residues before the disordered RGD region represented by magenta spheres (PDB ID 1D4M) [67]. The spacing between the RGD sites around an icosahedral fivefold axis is 60 A˚ for all three viruses. (a,b) Modified with permission from Ref. [59]. www.sciencedirect.com
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However, unlike EV1, these other picornaviruses interact with av integrins, which do not contain an I domain, and they interact via conventional integrin-binding RGD motifs. A striking feature of nonenveloped virus interactions with integrins is that this often involves very similar geometry/spacing of receptor engagement around the fivefold axis of the virion. The correspondence of the spacing ˚ ) of the five RGD loops around a single vertex of (60 A adenovirus with that of FMDV has been noted [59]. This is despite the fact that adenovirus and FMDV are structurally and evolutionarily distinct, and are from two different virus families, Adenoviridae and Picornaviridae. Less surprisingly FMDV and CAV9, both members of the picornavirus family, have a similar spacing for their integrinbinding RGD sites. The RGD sites of all three viruses ˚ and (adenovirus, FMDV and CAV9) are spaced by 60 A are found on mobile loops or peptide regions (Figure 4). Presumably this spacing is optimal for interaction of the virus with multiple integrins at a single capsid vertex. Perhaps the close packing of integrins induces a larger conformational change that is transmitted to the cytoplasmic integrin tails than would be induced by isolated RGD–integrin interactions. Conclusions and future perspectives It is not surprising that numerous viruses (as well as bacteria) have usurped integrins for cell invasion, because integrins are expressed on a wide variety of cells throughout the body. Moreover, integrin ligation by microbial pathogens elicits potent signaling responses that promote cytoskeletal reorganization and/or cell entry. Despite the obvious advantages of using integrins as entry receptors, viruses also use non-integrin receptors, such as members of the Ig superfamily (e.g. CAR and JAM-1). It is important to note that these receptors allow high-affinity binding of virions to cells but do not trigger cell signaling or promote entry. The different choices viruses make with respect to receptor usage may reflect the ‘trial and error’ nature of evolution or perhaps different requirements for virus replication in certain types of cells displaying integrins or non-integrin receptors. There are several key questions regarding virus–integrin interactions that have yet to be answered (Box 2). Both enveloped and nonenveloped viruses utilize integrins for cell invasion and they do so with a variety of mechanisms. One interesting observation that has emerged from recent investigations is that integrin avb3, in particular, which is known to exhibit substantial
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Box 2. Outstanding questions Does the precise spatial orientation of integrin-binding motifs on virus particles regulate cell signaling and infection/entry? What are the key signaling molecules that are common in the cellentry pathways of diverse viruses? Why are certain integrins used by a particular virus but not by others? Why is avb3 used by multiple virus families? Do integrins need to be in ‘active’ conformation for virus association and/or cell signaling? Are there novel integrin-binding sequences on viral proteins that remain to be discovered? Can integrin antagonists be made clinically useful for the treatment of viral infections?
diversity in ligand recognition [1], is one of the most comonly used receptors by members of diverse virus families (Table 1). It is, therefore, tempting to speculate that multiple viruses have evolved to take advantage of the promiscuous attributes of integrin avb3. Because integrins such as avb3 are capable of recognizing relatively small peptide sequences, this may provide a convenient pathway for virus evolution as integrin ligand sequences could be readily incorporated without drastically altering virus structure. Clearly the long co-evolution of viruses with their hosts has led to the variations on a theme that we are now discovering about virus–integrin interactions. Although much is understood now about integrin structure, questions still remain about the precise conformational changes that integrins must undergo to achieve both inside–out and outside–in signaling. The rapid progress made in recent years continues to aid our understanding of integrin structure. We anticipate that additional structural studies of virus–integrin complexes will help to elucidate the nature of the conformational changes induced by multivalent viral ligands and the diverse molecular mechanisms that can be involved in integrin signaling. Integrins also remain attractive drug targets for interfering with cell proliferation, migration or tissue localization of inflammatory, angiogenic and tumor cells [60,61]. Integrin-targeted drugs [62] might also modulate virus– ligand affinity and signaling, a situation that could prove useful in controlling infectious diseases. We anticipate that further investigation of virus–integrin interactions will contribute not only to a broader understanding of viral pathogenesis but also of integrin physiology. Such knowledge could eventually lead to the development of new antiviral as well as integrin-related therapeutic compounds.
Table 1. Integrins used by different viruses Virus family Adenovirus Herpesvirus Hantavirus Picornavirus
Reoviridae
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Virus type Adenovirus 2/5 HCMV HHV-8 SNV PHV EV1 FMDV CA9 EV9 Rotavirus Reovirus (Lang)
Integrins used avb1, avb3, aMb2, avb5 b1, avb3 a3b1, a2b1 allbb3, avb3 b1 a2b1 a5b1, avb3 avb3, avb6 avb3 axb2, a2b1, avb1, a4b7, avb3 b1
Role in infection Cell entry, signaling, endosome escape Cell entry, signaling Cell entry, signaling Attachment, entry (?) Attachment, entry (?) Attachment, signaling, entry Attachment, entry Attachment, entry Attachment, entry Attachment, entry Cell entry
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