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Abstract: Current understanding of the molecular basis of integrins as virus receptors has been achieved through over decades of study into the biology of the transmembrane glycoproteins and their interactions with several viruses. Over years, classical biochemical and physical analyses of integrins have helped to reveal the structure and function of some integrins, while knowledge gained by the study of the integrin-related viruses has helped define structures that are essential for virus adsorption and production of disease. Although many questions remain, the following review provides a summary of the current state of knowledge into the molecular basis of some viruses' interactions with related integrins, which are of potential practical significance because the rational design of drugs that inhibit virus-integrin interactions at the points of virus attachment or entry provides a novel approach to the therapeutic treatment of virus diseases.
Key words: Integrins; Ligands; Activation; Virus receptor
Integrins are a family of ubiquitous Î±/Î² heterodimeric glycoproteins that can interact extensively with ligands such as coagulation and fibrinolytic factors, complement proteins and cellular counter-receptors, epithelial and vascular matrix components in addition to viral proteins, which serves to link such diverse fields as hematology, neurobiology, thrombosis, cancer biology, developmental biology, inflammation viruses infection and even gene therapy (1-7). Many viruses such as Human coxsackievirus A9, Human papillomavirus, Adeno-associated virus 2 (AAV2), Hantaan virus, Foot-and-mouth disease virus, Human parechovirus1 and Human echovirus 1, 8, 9, 22 can initiate infection by attaching to activated integrins. This Review focuses on recent and selected advances relating to factors contribute to intergrin activation and the roles of integrin in viruses infection.
1. Structure of integrin
An integrin is about 280 Å long and consists of one Î± (150 to 180 kD) and one Î² (about 90 kD) subunit, both of which are type I membrane proteins. To date, there are at least 18 Î±ï¼ˆÎ±1~Î±11ã€Î±Dã€Î±Eã€Î±Lã€Î±Mã€Î±vã€Î±Xã€Î±IIbï¼‰ and 8 Î²ï¼ˆÎ²1~Î²8ï¼‰subunits which associate to form 24 different non-covalently bounded Î±/Î² heterodimers. The resulting heterodimers can then be grouped into subfamilies according to the identity of their Î± and Î² subunits or to ligand specificity (8). Each
integrin subunit is composed of large extracellular domains, a transmembrane region, and a short
Foundation item: the National Basic Research (973) Program (No.2005CB523201)
*Corresponding author: Chang Huiyun, Tel: 0931-8342052
cytoplasmic domain that in most cases consists of 20-70amino acid residues (except for Î²4 which contains a very large cytoplasmic domain of some 1000 amino acids). The structure between the Î± subunits is very similar, as do the Î² subunits. According to the identity of the Î± subunits, integrins
can be subdivided into 2 groups, one group with Î± subunitsï¼ˆÎ±1, Î±2, Î±10ï¼ŒÎ±11ï¼ŒÎ±L, Î±M and Î±Xï¼‰that contain an I ("inserted") domain(about 180 amino acids)which is a member of a family of von Willebrand A domains (VWA). Members of the other group (Î±3, Î±5, Î±6, Î±7, Î±8, Î±IIb, and Î±V) all share a post translational cleavage of their precursor into a heavy and a light chain. The light chain is composed of the cytoplasmic domain, the transmembrane region and a part of the extracellular domain (about 25 kD), while the heavy chain contains the rest on the extracellular domain (about 120 kD) (9). Nevertheless, all Î± subunits contain a Î²-propeller which constituted by 7 homologous repeats of 30-40 amino acids in their extracellular domain, spaced by stretches of 20-30 amino acids(Fig.1). The three or four repeats that are most extracellular contain sequences Asp-X-Asp-X-Asp-Gly-X-X-Asp or related sequences with cation-binding properties. More over, all the alpha subunits share the 5 amino acid motif GFFKR, which is located directly under the transmembrane region. The leg the of Î±-subunit consists of a thigh domain and two calf domains .Characteristic of Î² subunits is a four-fold repeat of cystine-rich segment believed to be internally disulfide bonded, and the N-terminal 40-50 KD is tightly folded with internal disulfide loops and contributes to the ligand-binding domain. The head of all Î²-subunits contains an I-like domain, which shares a common structure with Î±I domains (Fig. 1). The Î²I-like domain interacts with Î±-subunit, forming an interface for a ligand binding. The leg of Î²-subunit has a hybrid domain, plexin-semaphorin-integrin (PSI) domain, four cystein-rich repeats (I-EGF; epidermal growth-factor domains) and a novel cystatin-like fold (Î²-tail domain). A metal-ion binding site (MIDAS), essential for ligand binding is present in both the Î±I domain and Î²I-like domain. As for the Î±/Î² heterodimers, the N-terminal domains of Î± and Î² subunits combine to form a ligand-binding head which is connected by two stalks, each made up of one of the membrane-spanning segments and thus to the two cytoplasmic domains (1) (Fig.1). These cytoplasmic domains are believed to interact with cytoskeletal proteins and perhaps with other cytoplasmic components (10).
2. Integrin activation
Integrin activation initially refers to the changes required to enhance ligand-binding activity (the primary effector function of adhesion receptors), whereas activation of signalling receptors generally refers to the changes induced by ligand binding that enhance signal transduction (the primary effector function of signaling receptors). The finding that integrins also play important roles as signalling receptors serves to emphasize the importance of providing clear definitions of terms11ï¼‰.A general property of integrins is that they exist in at least two conformations, active (competent to bind ligand) and inactive (unable to bind ligand). Conversion from an inactive to an active state (integrin activation) is postulated to occur through two different mechanisms, collectively referred to as "inside-out signaling"; the first, affinity modulation, is mediated through conformational changes in the integrin ectodomain, whereas the second, avidity modulation, is mediated by clustering of heterodimers at the cell surface (12).
2.1 Integrin affinity
One important mechanism by which cells regulate integrin function is through tight spatial and temporal control of integrin affinity for extracellular ligands. This is achieved by rapid, reversible changes in the conformation of the extracellular domains of the integrin
Heterodimer (13).Transitions between these conformations have been shown to be regulated by both divalent cation occupancy and ligand binding(Fig. 2). Since integrins are conformationally flexible and contain a number of key hinge regions, exquisite changes in cationic environment regulate a complex pivoting of both Î± and Î² subunits about these hinges and the resulting alterations in shape have a direct effect on ligand-binding capacity. In general, Mn 2+ and Mg2+ usually promote ligand binding and Ca2+ usually has an inhibitory effectï¼ˆ14, 15ï¼‰.
2.2 Integrin clustering
Integrins differ from other cell-surface receptors in that they bind their ligands with a low affinity (106-109 liters/mole) and that they are usually present at 10-100 fold higher concentration on the cell surface. So only when after a certain stimuli or kinasesï¼ˆ16ï¼‰, these integrins cluster for example in focal contacts their combined weak affinities give rise to a spot on the cell surface which has enough adhesive (sticking) capacity to bind ligands. Clustering is considered to increase the avidity, but not the affinity, of molecular interactions, thereby increasing receptor occupancy by increasing the on-rate of binding (17).This occurs inside (for signalling and cytoskeletal proteins) and outside the cell (for ligands), and can be controlled bidirectionally. Thus binding of integrins to multivalent ligands in the extracellular matrix or on other cell surfaces causes accumulation of signalling complexes on the cytoplasmic face of the plasma membrane, and a wide selection of intracellular factors can induce formation of cytoskeletal and signalling complexes, which, in turn, recruit integrins via linker proteins.
3. Integrins and viruses infection
3.1General principles of virus-integrin interactions
Many viruses exploit the endocytic machinery of the host for invasion, in which, viruses must attach to the specific receptor(s) on the cell surface. These receptors can often have important functions in cell adhesion, cell-cell interactions, signalling and defence mechanisms. The binding of virus to a receptor can elicit changes in receptor conformation. These alterations may bring about signalling events that regulate both the viral entry process and the cellular response to the infection (18, 19). On the other hand, conformational changes in virus particles, triggered by receptor binding, can also facilitate virus entry and uncoating.
Integrins seem to be the "doors" for some viruses to enter the cell (Table 1). The interaction between a virus and integrins plays a key role in the virus multiplication cycle. This interaction brings about membrane permeabilization, fusion, and endocytosis. There are different complex strategies of integrin-dependent virus infectivity. Considering the roles integrins play, integrins can be used either as primary attachment receptors or as co-receptors in the entry process. When virus-integrin interaction considered, viruses are able to bind to integrins using pattern recognition sequences that are important for natural ligands (such as RGD , GRRP, LDV QAGDV) (20), or interact with unique regions of integrins without necessarily having a recognition sequence.
3.2 Viruses that utilize integrins as receptors
The RGD-binding integrins are among the most promiscuous in the family, several viruses have been reported to utilize RGD-dependent integrins to initiate infection. For example, Adenovirus has been shown to interact with Î±vÎ²3, Î±vÎ²5, and Î±vÎ²1 integrins by virtue of a high-affinity arginine-glycineaspartate (RGD) domain present in the penton bases of the capsids (21) and human parechovirus type 1 uses Î±vÎ²3 and Î±vÎ²1,whereas coxsackievirus A9 has been shown to use Î±vÎ²3 (22). In addition, Î±vÎ²1, Î±vÎ²3, and Î±5Î²1 have been implicated as receptors for coxsackievirus A9, the Barty strain of echovirus type 9, and adenovirus, respectively. Integrins have also been implicated as receptors for rotaviruses and papillomaviruses. In addition, Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8) uses Î²1 integrin for infectionï¼Œrecently, Dyson and colleagues determined that RGD peptides, antibodies, and siRNA specific to 1 integrins significantly lowered the ability of the primary effusion lymphoma supernatants to induce tubule formation by endothelial cells, however, the 1 integrins did not seem to have a major role in cellular attachmentï¼ˆ23ï¼‰.
Nevertheless, some virus-interactions are independent of RGD sequence. For instance, cytomegalovirus can interaction with integrin via a highly conserved disintegrin-like
domainï¼ˆ24ï¼‰.In addition, Hantaviruses, a significant human pathogen that causes severe respiratory disease, have also been reported to use integrin Î±3Î²3 for infection, but this interaction seems to be independent of the RGD motif (25).In the case of AAV2, which lacks this RGD motif, integrin Î±vÎ²5 has been identified as a coreceptor for cellular entry. However, Aravind et al recently demonstrate that a highly conserved domain that contains an asparagine-glycinearginine (NGR) motif on the VP3 domain of the AAV2 can bind integrin Î±5Î²1 with moderate affinity, thus mediate AAV2 infection in human embryonic kidney 293 cellsï¼ˆ26ï¼‰.There is also the hypothesis that bacteriophages (bacterial viruses) use a cellular receptor Î²3 integrins for their attachment to eukaryotic cells. Some phages (e.g., T4) present a KGD sequence in their external proteins (there are 55 copies of the KGD motif in the head corner protein of the T4 phage) (27).
3.3 Role of adaptor proteins and kinases in Integrin-mediated viruses infection
The cytoplasmic domains of integrins, particularly -subunits, interact with adaptor proteins (e.g., vinculin, talin, and Î±-actinin) that are critical for transmitting mechanical force between the ECM and the actin cytoskeletonï¼Œin addition, integrins and their associated adaptor proteins recruit signaling molecules such as focal adhesion kinase, proline-rich tyrosine kinase 2, Src, c-Src kinase, Integrin-linked kinase (ILK), the protein kinases GSK-3, protein kinase C (PKC), and p21-activated kinase (PAK) to the membrane. Kinases have been known for some time to regulate endocytosis, and recently an unexpectedly large number of kinases were shown to regulate clathrin- and caveolin-mediated endocytoses (28). Increasing evidence suggests that small GTPases such as Arf6 and members of the Rab family control integrin internalization and recycling back to the plasma membrane along microtubules. Several Kinases that have well-established roles in integrin signaling can move integrins from the back to the front of migrating cellsï¼Œthus facilitate virus adsorption(29, 30). For instance, Echovirus1 and rotavirus enter cells via Î±2Î²1 integrin-mediated endocytosis, regulated by dynamin-dependent mechanisms (31). Adenoviral entry via Î±V integrin is dependent on GTPase Rab5 (32). The rotavirus spike protein Vp4 is able to bind both to the extracellular domain of Î±2 subunit and to Rab5 in the cytosol (33).More targets downstream of these kinases are likely to be identified, and many may be proteins that function in other activities involving virus endocytosis. Identifying these will thus provide insight into how different endosomal pathways integrate extracellular signals and coordinate their activities in viruses infection.
The growing number of viruses that use integrins for cell entry suggests that these receptors are not only convenient portals for cell entry, but also have more significant functions. Integrin ligation increases the kinetics of pathogen virus internalization into cells, a situation that would prevent virus particles from being rapidly inactivated by antibodies and/or complement. Integrin signaling could also provide a more stable environment for the microbe once it has established the infection by down-regulating the host immune response. Microbes that use Î²1 integrins on cells of the immune system also provide an opportunity for establishing a latent infection in these cells, as well as facilitating pathogen dissemination throughout the host. Microbial pathogen spread could also be enhanced through integrin-mediated cell migration. However, pathogen entry is a complicated process that is also regulated by enzymes and signaling molecules secreted by the
pathogen, further studies with viruses that are relate to integrins and kinase should help to increase our understanding of the precise function of integrin in cell entry and microbial pathogenesis.
We thank Humphries for permission to use his schematic model of integrin structure and Enya for his constructive criticism of the manuscript. The work from my laboratory discussed within the article is supported by National Basic Research (973) Program (No.2005CB523201). Apologies to those authors whose work we could not cite due to space restrictions.