JOURNAL OF VIROLOGY, Sept. 2005, p. 10839–10851 Vol. 79, No. 170022-538X/05/$08.00�0 doi:10.1128/JVI.79.17.10839–10851.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Tetraspanins in Viral Infections: a Fundamental Role in Viral Biology?F. Martin,1 D. M. Roth,2,3 D. A. Jans,2 C. W. Pouton,3 L. J. Partridge,4 P. N. Monk,1 and

G. W. Moseley2*Academic Neurology Unit, Division of Genomic Medicine, University of Sheffield, Sheffield, United Kingdom,1 Department ofBiochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia,2 Victorian College of Pharmacy,

Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia,3 and Department of Molecular Biology andBiotechnology, University of Sheffield, Sheffield, United Kingdom4

The tetraspanins are a broadly expressed superfamily oftransmembrane glycoproteins with over 30 members found inhumans and with homologues conserved through distantly re-lated species, including insects, sponges, and fungi. Membersof this family appear to form large integrated signaling com-plexes or tetraspanin-enriched microdomains (TEMs) by theirassociation with a variety of transmembrane and intracellularsignaling/cytoskeletal proteins (49). These interactions link tet-raspanins to an array of physiological functions and, in conse-quence, to numerous endogenous pathologies, including can-cer development and inherited disorders (Table 1).

Tetraspanins are also known to have roles in the pathologyof infectious diseases such as diphtheria, malaria, and numer-ous viral infections (Table 1). The literature currently indicatesthat specific tetraspanin family members are selectively asso-ciated with specific viruses and affect multiple stages of infec-tivity, from initial cellular attachment to syncytium formationand viral particle release. Thus, the relationship of tetraspaninswith viruses appears to be particularly complex.

Here, we will consider this data in the context of recentdevelopments in tetraspanin biology, particularly in our under-standing of the architecture and function of TEMs. With thebenefit of recent insights into tetraspanin function in cell fu-sion events and intracellular trafficking, we discuss commonfeatures of tetraspanin/viral associations which indicate a fun-damental role for TEMs in a number of viral infections. Wewill also consider the existing therapeutic strategies for humanimmunodeficiency virus (HIV), hepatitis C virus (HCV), andhuman T-cell lymphotropic virus type 1 (HTLV-1), focusing onthe potential therapeutic value of targeting TEMs, using pep-tide reagents based on tetraspanin extracellular regions.

THE TETRASPANIN SUPERFAMILY

Structural features of the tetraspanin superfamily. Tet-raspanins are type III membrane glycoproteins which span theplasma membrane four times, producing two extracellularloops and short intracellular regions (Fig. 1). A defining struc-

tural signature of the superfamily is the “tetraspanin fold” ofthe larger extracellular loop (LEL). In this region, disulfidebonding of four absolutely conserved cysteines forms a subloopstructure containing a region which is hypervariable betweenfamily members and between species homologues of the sametetraspanin (Fig. 1) (81, 142). The cysteines are present inthree variously conserved motifs (CysCysGly, ProXSerCys[where X � any amino acid], and GluGlyCys) (Fig. 1), whereinthe flexibility and constraint imparted by the conserved Glyresidue in CysCysGly and by Pro in ProXSerCys contribute tosubloop formation (68). Many members of the tetraspaninfamily have one or two additional pairs of Cys residues in thetetraspanin fold region, potentially allowing the formation ofcomplex subloop structures. The region of the LEL outside ofthis subloop shows greater structural conservation, formingthree �-helices which not only form a structural platform topresent the tetraspanin fold but may also contribute indepen-dently to tetraspanin function (see below) (68, 152).

Conservation of the canonical LEL Cys, Gly, and otherresidues, including charged transmembrane amino-acids, dis-tinguishes members of the tetraspanin superfamily properfrom other “tetraspan” proteins (168). Modification of tet-raspanins includes O- and N-linked glycosylation. N glycosyl-ation sites are largely located in the LEL, and glycosylation hasbeen shown to regulate specific tetraspanin functions (111). Nglycosylation is generally assumed to occur at classical AsnXSer/Thr sites, but the LELs of some tetraspanins, includingCD9 and CD81, contain the rarer AsnXCys N-glycosylationmotif (138) which, interestingly, incorporates canonical cys-teines. Mutation of the only AsnXSer sites in CD9 does notprevent N glycosylation, indicating that the AsnXCys site maybe utilized in some cases (G. W. Moseley, L. J. Partridge, andP. N. Monk, unpublished data). Palmitoylation of tetraspaninshas also been shown to be functionally significant, as it appearsto participate in the formation of heterotetraspanin associa-tions (14) and to regulate association with lipid rafts (seebelow) (16).

Tetraspanin-enriched microdomains. Tetraspanins affectmultiple events in vitro, including cellular signaling, migration,adhesion, fusion, cytoskeletal reorganization, and prolifera-tion, which appear relevant to altered expression patterns seenduring cellular activation, differentiation, proliferation, andmalignant transformation in vivo (reviewed in references 87and 168). Tetraspanins are expressed by all mammalian tissues,

* Corresponding author. Mailing address: Nuclear Signalling Labo-ratory, Department of Biochemistry and Molecular Biology, MonashUniversity, Building 13D, Monash, Victoria 3080, Australia. Phone:61-3-99051220. Fax: 61-3-99053726. E-mail: greg.moseley@med.monash.edu.au.

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although the complement of family members expressed is tis-sue specific (Table 1). It is unsurprising, therefore, that tet-raspanin functions have been identified in an array of celltypes, including platelets, epithelial/endothelial cells, musclecells, and photoreceptor cells and cells of the immune, centralnervous, and reproductive systems (87, 99). The basis for thisbroad functionality appears to be the capacity of tetraspaninsto form multiple intermolecular interactions with a restrictedbut varied complement of transmembrane and intracellularmolecules (Fig. 2).

Tetraspanins have no intrinsic enzymatic activity or typicalsignaling motifs (with the exception of the recently identified14-3-3 binding motif in CD81 [20]), so it has been predicted thatthey act primarily as novel adapter proteins, facilitating the inter-action of associated molecules in tetraspanin signaling networks

(tetraspanin web [135] or TEMs [49]). The evidence that phys-ical associations of tetraspanins are functionally relevant iscompelling. For example, in vitro modulation of tetraspan-ins on cell adhesion/migration or growth have been shown tobe dependent on tetraspanin-associated integrins (76, 93,143) and progrowth factors (75, 144). Similar observationshave been made in vivo in tetraspanin-null animals (167).

Analogies have been drawn between TEMs and lipid rafts.In the latter, protein-protein interactions are facilitated byassociation with membrane regions enriched for cholesteroland glycosphingolipids (149). In either lipid rafts or TEMs theassociated proteins are believed to form large, integrated sig-naling platforms. Although TEMs and lipid rafts exist as sep-arate entities, they have been shown to interact physically andfunctionally (17, 27).

TABLE 1. Members of the tetraspanin superfamily with reported links to pathologiesa

Tetraspanin Alternative name Tissue distribution Pathological link Reference

CD9 TM4SF2, DRAP-27, MRP, MIC3,p24

Platelets, early B cells, activated anddifferentiating B cells, activated Tcells, eosinophils, basophils,endothelial cells, megakaryocytes,epithelia, dendritic cells, brainand peripheral nerves, fibroblasts,lung, kidney, liver, vascularsmooth muscle, skeletal muscle,keratinocytes

Expressed on leukemias,melanomas, and cancers ofbreast, lung, colon, and pancreas;modulates affinity of diphtheriatoxin receptor (pro-HB EGF);linked with FIV, HIV, and CDV(see text)

12, 150,168

CD63 TM4SF1, MEL1, ME491,granulophysin, LAMP3, OMA81H,MLA1

Wide lymphoid and nonlymphoiddistribution, including platelets,neutrophils, monocytes,macrophages, dendritic cells,endothelia, megakaryocytes,epithelia, fibroblasts, lung, kidney,liver, smooth muscle, skeletalmuscle, peripheral nerves,pancreas, and cardiac muscle

Expression correlated withmelanoma progression;b linkedwith HIV infection (see text)

12, 150,168

CD81 TM4SF10, TAPA-1 Broad expression on nonlymphoidtissues and on lymphocytes,thymocytes, follicular dendriticcells, eosinophils, monocytes, andepithelia

Potential roles in HCV infection(see text); involved ininternalization of Plasmodiumfalciparum and P. yoelii

81, 64,147

CD82 TM4SF11, Kangail, KAI1, SDT6,R2 Ag

B and T cells, NK cells, monocytes,granulocytes, and platelets andvarious nonhemopoietic cells

Expression linked to prostate, lung,colon, hepatoma, and breastcancers; involved in HTLV-1infection (see text)

12, 74

CD151 TM4SF32, PETA3, SFA1, gp27 Endothelial cells, platelets, dendriticcells, megakaryocytes, epithelia,lung, kidney, liver, smooth andskeletal muscles and peripheralnerves, keratinocytes, pancreas,and cardiac muscle

Upregulated in HTLV-1 infection;expressed in colon and lungcancers

5, 150

CD231 TM4SF27, A15, TALLA1, TM4-2b Heart, brain, lung, liver, skeletalmuscle, kidney, pancreas, andimmature T cells.

Expressed in neuroblastomas andleukemias; gene inactivated inX-linked mental retardation

174, 168

Rom1 TM4SF28 Photoreceptor cells Retinis pigmentosa 42

Peripherin TM4SF29, RDS, RP7 Photoreceptor cells Retinis pigmentosa 42

a Additional tetraspanins which have been linked to tumor development include NET-1 (cervical cancer [165]), C0-029 (colorectal carcinomas [56]), and SAS(sarcomas [63]).

b There are numerous reports correlating CD63 epitope expression with melanoma progression, but the precise nature of this relationship is controversial (104, 167).

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Architecture of TEMs. Recent studies assessing the strength/proximity of tetraspanin interactions have indicated that TEMsare constructed from a hierarchy of interactions (Fig. 2) (49,167). Primary (or high-order) tetraspanin interactions gener-ally appear to be mediated by the LEL region, particularly thesubloop and proximal regions, a notable exception being ho-motypic tetraspanin associations, which are suggested to bemediated by regions of the LEL away from the subloop (Fig. 1)(152). These are the most robust of TEM interactions, beingstable in relatively hydrophobic detergents such as TritonX-100 and incorporating significant proportions of the cellularcomplement of the associated proteins. The fact that they canbe stabilized by short covalent chemical cross-linkers indicatesthat they involve direct extracellular interactions.

Secondary interactions involve the transmembrane and/orintracellular regions (Fig. 1) (10, 14). These interactions areless stable in high-stringency detergents but are maintained inmilder conditions (Brij-96, Brij-97) and involve tetraspanin-tetraspanin associations that show some dependency on pal-mitoylation (14). These interactions are thought to indirectlylink primary complexes and thereby facilitate their cross talk.

Tertiary, low-order interactions are stable only in low-strin-gency detergents (CHAPS, Brij-99). While higher order inter-actions are isolated as discrete entities, tertiary interactions are

large complexes containing multiple tetraspanins and associ-ated molecules. Thus, tertiary interactions are hypothesized toresult from the large-scale coalescence of higher order com-plexes. The study of tertiary interactions is complicated bytheir poor stability and extensive nature, meaning that thecomposition of the complexes is less well defined than forhigher order associations. The use of low-stringency detergentsto isolate these complexes suggests the possibility of nonspe-cific capture of molecules in insoluble micelles. However, sev-eral tertiary interactions appear to be functionally relevant orhave been confirmed by different methods, including fluores-cence resonance energy transfer (154). Importantly, Claas etal. (18) have demonstrated that tertiary interactions can existindependently of lipid rafts/large detergent-insoluble fractions.

Conceptually, TEMs may form extended structures of pri-mary tetraspanin complexes indirectly connected in large con-tiguous networks (Fig. 2). However, it is unlikely that TEMsare static structures, and the differing properties of primaryinteractions compared with those of lower order interactionsmay relate, at least in part, to a more transitory nature of thelower order interactions, not only to their spatial arrangement.The localization of tetraspanin complexes, subcellularly and inthe plane of the membrane, is known to change in response tocertain stimuli, as does the molecular composition of isolated

FIG. 1. The tetraspanin CD81. The primary sequence of CD81 (single-letter amino acid code) is shown schematically in the context of itstransmembrane architecture. Key conserved residues in the tetraspanin superfamily include charged transmembrane residues and canonicalcysteines which are disulfide bonded to form a functional subloop in the LEL. Sites of potential modification by N-glycans and palmitoylation areshown, and approximate locations of sites of molecular associations (homodimerization/heterodimerization of tetraspanins and primary interactionwith nontetraspanin proteins) are highlighted. CD81 lacks classical NXS/T glycosylation sites commonly found in other tetraspanins and a broadlyconserved E/Q residue in the fourth transmembrane domain. The four residues which differ between human CD81 LEL and African green monkey(T163, F186, E188, D196) CD81 LEL are shown. PKC, protein kinase C.

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complexes (59, 118). The dynamic subcellular localization oftetraspanins has been suggested to relate to roles in traffickingof web-associated molecules or as novel chaperone proteins inintegrin maturation (9, 33, 60, 89, 134, 173). Moreover, in Bcells the CD81/CD21/CD19 coreceptor complex interacts dy-namically with lipid rafts, modulated by palmitoylation ofCD81 (16), and regulated association of CD82, lipid rafts, andthe cytoskeleton has been reported (27). Cytoskeletal associa-tion of TEMs is likely to be involved in their subcellular/membrane distribution. Thus, TEMs, like lipid rafts, are likelyto be highly dynamic and regulable structures.

Tetraspanin function in vivo. A major feature of the tet-raspanin web hypothesis (135) was that it provided an expla-nation for the similar functional effects of different members ofthe superfamily observed in vitro. This hypothesis has receivedsupport from studies of tetraspanin-null mice, where similar

effects of knockouts of CD151, CD81, CD37, Tssc-6 have beenseen in T cells (167) and in the CD9-knockout mouse, whereCD81 expression in vitro can compensate for deficiencies insperm-egg fusion (65). Moreover, tetraspanin-knockout miceare largely healthy, generally displaying only mild phenotypicabnormalities. This is surprising, as quite dramatic in vivophenotypes result from mutation of the primary partners ofsome tetraspanins (69, 166). Thus, it appears that there is acertain degree of functional redundancy between tetraspanins.

While single-tetraspanin knockouts display mild phenotypes,phenotypic abnormalities are widespread and are found in Tcells, platelets, and keratinocytes of CD151-null mice (77, 166),in the central nervous system, B and T cells, and retinal pig-ment epithelial cells of CD81-null mice (39, 100, 151), and ingametes, smooth muscle cells, and the peripheral nervous sys-tem of CD9-null mice (58, 99, 140). It would appear likely,

FIG. 2. Possible architecture of the tetraspanin web. The principal interactions of TEMs are robust primary interactions which involve directextracellular tetraspanin-protein interactions. Secondary interactions are less robust and include heterotetraspanin interactions which facilitateindirect interaction of primary tetraspanin complex proteins. Tertiary interactions are the least-robust complexes and are thought to reflectlarge-scale coalescence of the higher order interactions into large signaling domains. Based on the known structure of the tetraspanin-richurothelial plaque, this order of interactions is shown as interlinked hexagons (98). Interactions within TEMs include homotypic and heterotypictetraspanin associations and association with adhesion molecules (integrins [9]), immune cell receptors (e.g., Fc receptors and T-cell receptor(TCR), CD4/CD8, and MHC proteins [103, 167]), growth factors and their receptors (pro-HB-EGF [136], pro-transforming growth factor � [144],and epidermal growth factor receptor [110]), intracellular signaling molecules (protein kinases hck, lyn, and PKC [167, 176] and phosphatidyl-inositol 4-kinase [172]), and cytoskeletal components (27), as well as members of a novel transmembrane immunoglobulin superfamily (EWI-2,EWI-F) (152).

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therefore, that TEMs per se have important roles in the phys-iology of most, if not all, cell types. The removal of a singletetraspanin shows some redundancy, owing to the extensivenature of TEMs and the expression of close homologues. How-ever, the clear phenotypic abnormalities observed indicate thatnot all functions of a single tetraspanin can be replicated bytheir counterparts (as is suggested by the evolutionary conser-vation of the members of this large family). The production ofknockout mice lacking multiple tetraspanins may lead to adelineation of the overlapping and unique functions of evolu-tionarily close members of the tetraspanin family.

As described above, studies of the structure of TEMs haverelied heavily on detergent solubility studies, which are clearlylimited and relatively insensitive. Similarly, with the data cur-rently available, the functional/physical relationship of tet-raspanin associations remains inferred, as it has not formallybeen correlated through mutagenesis of tetraspanins. The re-expression of mutated tetraspanins in null mice may thereforepermit more intricate and physiologically relevant investiga-tions of the structure and functional architecture of TEMs.

TETRASPANINS IN VIRAL INFECTIONS

Given the broad physiological importance of tetraspanins, itis not surprising that tetraspanins have been commonly iden-tified in cellular disorders (Table 1). There is increasing evi-dence for the involvement of tetraspanins in infections by var-ious microbes, but they have been most commonly identified asplaying a role in viral infections. The remainder of this reviewwill focus on these associations.

FLAVIVIRUSES: HCV AND CD81

Interaction of CD81 and HCV-E2. The most exhaustivelydocumented involvement of a tetraspanin in viral infection isof CD81 in HCV infection. HCV is a small, enveloped RNAvirus whose genome encodes a single polyprotein that is pro-cessed by viral/cellular proteases into structural and nonstruc-tural proteins. Structural proteins include the envelope glyco-proteins E1 and E2, which mediate viral binding and entry intohost cells (78).

CD81 was the first protein ligand identified for HCV, spe-cifically for E2 protein. The interaction was identified using acDNA library from a subclone of a human T-cell lymphomaline with high E2 binding capacity and was confirmed by thedemonstration that monoclonal antibodies to CD81-LEL caninhibit the binding of recombinant E2 to Epstein-Barr virus-transformed B cells (122) and that human CD81-LEL specif-ically binds E2 from HCV-infected plasma. The chimpanzee isthe only species other than humans that shows susceptibility toHCV (132), and vaccination with E2 glycoproteins can protectchimps from the virus. The physiological significance ofCD81/E2 association was implied by the demonstration thatthis protection correlated with the ability of sera to block theirinteraction (122).

The sequence in the LEL essential for binding E2 is con-tained within residues 164 to 201, a region that forms theepitope for antibody 1D6, which inhibits E2 binding to CD81(Fig. 1) (50). Inhibition of E2 binding by a small peptideanalogue of CD81 LEL has highlighted the role of residues 176

to 189 (30). African green monkey (AGM; Cercopithecus ae-thiops) CD81 differs from human CD81 LEL by only fourresidues (Fig. 1), but AGM-CD81 is unable to bind to E2 andAGMs are not susceptible to HCV. Reciprocal mutations ofhuman and AGM CD81 have shown that the key residue inthis interaction is CD81 Phe186 (50). The importance ofPhe186 was confirmed when tamarin monkey (Saguinus im-perator) CD81 was shown to be able to bind to E2 with a higheraffinity than human CD81 (1). Phe186 is conserved in tamarinCD81, and mutation of this residue can inhibit E2 binding (97).Mutational analysis has determined that the E2 binding site onCD81 also comprises residues Ile182, Asn184, and Leu162,forming a hydrophobic E2 binding surface of approximately806 Å2 (31). It has also been suggested that the SEL domain isimportant during HCV infection, although this appears to re-late primarily to its role in promoting surface expression ofCD81 by masking an intracellular retention signal (91).

E2 binds to cells of human origin but not to cell lines derivedfrom AGM, rat, and rabbit (36). Thus, the interaction appearsto be dependent on human (h)CD81 and not to require othermolecules in human cells, as rat cell lines can bind E2 proteinwhen transfected with hCD81. Binding to this particular tet-raspanin is also specific, as E2 does not bind to hCD9-, hCD63-or hCD151-transfected rat cells (36).

Based on this evidence, it has been suggested that CD81 maybe a cellular receptor for HCV. However, recent data indicatesthat CD81 is not sufficient for HCV entry, and the relevance ofthe CD81-HCV E2 association for viral entry remains unclear.One study has reported that binding of HCV to three hepato-cyte-derived cell lines was not blocked by anti-CD81 antibody(137). It has also been shown that hepatocarcinoma cells thathave lost the expression of CD81 mRNA still show a small butdetectable level of HCV binding (120). Tamarins are not sus-ceptible to HCV infection, but tamarin CD81 binds to E2 withgreat affinity (1), while hCD81 transgenic mice cannot be in-fected with HCV (91) and certain human liver cell lines arenonpermissive to infection by HCV-HIV pseudotypes despiteCD81 expression. Thus, it appears that formation of theCD81-E2 complex does not necessarily confer susceptibility tothe virus. It has also been observed that different HCV strainsutilize CD81 to different extents (70, 94, 131); E2 subtype 1b,for instance, shows a lower binding affinity to CD81-LEL thanE2 subtype 1a. The CD81-LEL Phe186Leu mutation has beenshown to abolish the binding of CD81 LEL to soluble E2 strainH but not to strain Con 1, even though this mutation has noeffect on the ability of transfected HepG2 cells to supportinfection by pseudotype viruses expressing either Con 1 orstrain H E2 (175). Another study using a more diverse range ofHCV glycoproteins revealed that the CD81-LEL Asp196Glumutation reduced the infectivity of HCV pseudotypes express-ing CH35, HJC4, and C6a1 glycoproteins by 70 to 96% (94).These data suggest that all HCV pseudotypes require CD81for infection of HepG2 cells but that the importance of theinteractions between CD81 LEL and HCV glycoproteins dif-fers between viral strains. The available evidence suggests thatCD81 represents an important component in HCV cellularreceptor activity but that this process is complex and likely toinvolve multiple receptor complexes. Further research will berequired to establish the in vivo relevance of these in vitroobservations.

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CD81 in viral entry and release. There are other candidatereceptors for HCV, namely the scavenger receptor class B type1 (SR-B1) (139), dendritic cell-specific intracellular adhesionmolecule 3-grabbing nonintegrin (DC-SIGN) (84, 126), andlow-density lipoprotein receptor (LDLR) (170). One study hasestablished a requirement for both CD81 and SR-B1 for bind-ing and infectivity of HCV pseudoviruses, indicating a role forCD81 as a coreceptor in pseudotype infections (8). Similarly,pseudotype infections can be inhibited or enhanced by CD81silencing or expression, respectively (175), indicating a role forCD81 in viral entry. However, CD81 alone may be insufficientfor HCV entry, as the antibody-mediated internalization rateof CD81 is extremely low compared with that of other surfaceproteins such as CD4 and CD71 (120). Chimeras consisting ofCD81 and the cytoplasmic domains of two receptors that fre-quently undergo endocytosis (LDLR and the transferrin re-ceptor [TfR]) have been constructed (156); both chimeras sig-nificantly increase the endocytosis of recombinant HCV-E2protein, anti-CD81 antibody, and E2-enveloped viral particlesfrom the serum of HCV-infected patients. Expression of thechimeras in the Huh7 hepatoma cell line also resulted in in-creased viral replication, suggesting that additional factors arerequired for efficient endocytosis of wild-type CD81/virus com-plexes. Thus the mechanism of receptor-HCV complex inter-nalization is still poorly understood. Some lines of in vitroevidence indicate the involvement of receptor complexes in-corporating CD81, but further research will be required toidentify additional receptor components which contribute toefficient endocytosis

As discussed above, TEMs may have a general role in reg-ulating the trafficking of proteins associated with tetraspanins.There is precedent of a pathogenic exploitation of these pro-cesses: CD81 has been linked to entry of plasmodium intohepatocytes in the context of a parasitophorus vacuole (147).Thus, it is possible that viral proteins which interact with tet-raspanins become incorporated into TEMs, facilitating theirtransport into or out of the cell. In the absence of humanCD81, mammalian cells transfected with HCV E1-E2 cDNAalmost completely retain these proteins in the endoplasmicreticulum. However, when cotransfected with hCD81, a smallfraction of these proteins pass through the Golgi and are foundassociated with extracellular exosomes (92). The CD81Asp196Glu mutation decreases the susceptibility to infection(see above), despite having only a minimal effect on binding ofE2. It is possible that this mutation decreases infectivity byaltering the intracellular trafficking characteristics of CD81(50), lending further support to the notion that CD81-E2 in-teractions are involved in facilitating trafficking of HCV par-ticles within the cell. Thus, CD81 may play a role in facilitatingviral glycoprotein translocation into exosomes during viral exitand not exclusively in cell entry.

The possible involvement of CD81 in facilitating vacuoletrafficking/fusion/release is consistent with hypothesized rolesfor other tetraspanins in physiological processes (167, 169) andsimilar to events described for the role of CD63 in HIV-1infection (see below). In vitro studies showing that anti-CD81antibody delayed fusion of a mouse myogenic cell line supporta role for CD81 in membrane fusion events (155). Anti-CD9antibody induced the same effect, suggesting that TEMs con-taining different tetraspanins may be involved in this event.

Further evidence for TEM activity includes the demonstrationthat antibodies to CD9 or CD81 delayed syncytia formation ina rhabdomyosarcoma cell line (155). CD9 is also involved insperm-egg fusion (15, 79, 99), and significantly, the impairedfusion seen in CD9-null animals can be compensated in vitroby CD81 expression (65). Also, recent data suggest that solubleLEL domains of both CD9 and CD63 can inhibit concanavalinA-induced fusion of human blood monocytes, suggesting that arange of tetraspanins may be involved in the mechanism of cellfusion (V. Parthasarathy, G. W. Moseley, A. Higginbottom,P. N. Monk, R. C. Read, and L. J. Partridge, unpublisheddata). Therefore, TEMs containing multiple tetraspanins areinvolved in membrane fusion events which are likely to relateto roles in viral infection.

Potential roles of CD81 and E2 in modulation of the im-mune response. Although HCV-infected patients show bothcellular and humoral immune responses, over 85% are unableto eliminate the virus. In common with other leukocyte-ex-pressed tetraspanins, CD81 has roles in modulating immunecell function (17, 28, 86). An intriguing possibility is that theinteraction of CD81 with E2 could modify immune responsesin HCV-infected individuals. It has been shown that cross-linking of natural killer (NK) CD81 with immobilized E2 in-hibits NK cell response to interleukins-2, -12 and -15, whichmay affect early innate immune response to HCV infections(21, 159), while E2 cross-linking of CD81 on B- and T-cell linesalters proliferation and aggregation (36). E2 engagement ofCD81 also causes the secretion of the chemokine RANTES byCD8� T cells and the subsequent down-regulation of its re-ceptor, CCR5, a mechanism by which HCV might interferewith lymphocyte migration (109). These events may providestrategies for immune evasion or, alternatively, may relate tothe immune dysregulation effects of chronic HCV infections,including type 2 diabetes mellitus, glomerulonephritis, arthri-tis, and non-Hodgkin’s lymphoma (157). As CD81 is widelyexpressed and involved in an array of functions, there are anumber of potential biological effects of E2-CD81 interaction.Flint et al. (36) hypothesized that interactions between E2 oninfected hepatocytes and CD81 on uninfected cells couldprime the latter for infection. These are certainly intriguingpossibilities, and the genuine physiological significance of thebiological effects of E2 interactions with CD81 awaits furtherresearch.

RETROVIRUSES: HTLV-1, HIV, AND FIV

HTLV-1. HTLV-1 is a type C retrovirus that infects CD4�

lymphocytes in vivo (130), although CD8 T cells may also serveas a reservoir (107). This tropism contrasts with the in vitroability of viral Env protein to bind and enter multiple cell types(13, 45, 67, 153, 158). The receptor for HTLV-1 is unknown,but a likely candidate is the human GLUT1 glucose trans-porter (88). Molecules involved in syncytium formation, virusadsorption, and infection include ICAM-1, ICAM-3,VCAM-1, and tetraspanins (22, 52, 123, 124). Cell-cell fusionvia syncytium formation is thought to be one of the maintransmission mechanisms of HTLV-1 (19). The tetraspaninCD82 was identified as an antigen recognized by several anti-bodies found to inhibit HTLV-1-mediated syncytium forma-tion (38, 57). Coexpression of CD82 with HTLV-1 Env glyco-

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protein in COS-1 cells had an inhibitory effect on syncytiumformation and HTLV-1 infectivity, although the incorporationof Env into nascent virions was unaffected (124).

CD82 is expressed on the surfaces of most types of periph-eral blood mononuclear cells (PBMCs) and granulocytes, al-though CD16� NK cells show very low levels of expression andCD4� T cells express higher levels than CD8� T cells (57).CD82 is heterogeneously glycosylated in T cells, with apparentmolecular mass ranging from 35 to 50 kDa (57). Activation ofT cells (e.g., using phytohemagglutinin) and/or infection withHTLV-1 results in up-regulation of CD82 expression and alsoincreases the amount of glycosylation (38, 57). The glycosyla-tion state of CD82 has been shown to be essential for itscorrect functioning (111, 112), possibly by altering the affinityof CD82 for membrane proteins such as integrins. Coimmu-noprecipitation experiments show that HTLV-1 Env glycopro-teins interact with highly glycosylated forms of CD82, althoughinteractions with immature forms of the tetraspanin suggestthat these interactions occur early in the secretory pathway(124). The tetraspanin CD151 has also been shown to be up-regulated in HTLV-1-infected cells (47, 48), and this has beenlinked to increased adhesion of infected cells to the extracel-lular matrix. CD82 also appears to be involved in cellularadhesion and motility events (112), and it is possible thatCD151 and CD82 have common functions during HTLV-1infection. CD82 has recently been proposed to act as an adap-tor protein, linking lipid rafts and the actin cytoskeleton in Tcells (26). In this role, it is likely to control the function ofmolecules such as ICAM-1 and �1 integrins that are directlyinvolved in the cell-cell interactions that lead to syncytiumformation (22). Thus, although CD82 does not act as a cellularreceptor for HTLV-1 or affect expression levels of HTLV-1proteins, it is likely to function in concert with CD151 andother tetraspanins in TEM-dependent cell fusion events.

HIV-1. Entry of the retrovirus HIV (genus, Lentivirus) intocells occurs in three distinct steps: the attachment of virions tocell surface CD4� by envelope gp120 protein, the subsequentinterplay of the conformationally altered gp120 with cellularCCR5 and CXR4 coreceptors, and, finally, envelope gp41 pro-tein-mediated fusion to target cells (102). The HIV genomeconsists of two copies of RNA which are reverse transcribed toDNA to form the preintegration complex containing the viralmatrix (MA), integrase, and Vpr proteins as well as severalhost proteins, which is imported into the nucleus and incorpo-rated into the host chromosomes, where it can become latentfor 3 to 10 years (44). The subtypes of HIV, HIV-1 and HIV-2,have the same modes of transmission and are associated withsimilar opportunistic infections, but immunodeficiency seemsto develop more slowly with HIV-2 (90, 148).

A role for the tetraspanin CD63 in HIV-1 infection was firstsuggested when it was shown to be up-regulated from intracel-lular vesicles to the surfaces of HIV-1-infected cells and selec-tively incorporated into budding structures and newly synthe-sized virus particles (95, 96). Consistent with a role in viralrelease, vesicle fractions in infected H9 T cells contain in-creased levels of CD63 compared with noninfected cells (41).

Macrophages are sources of virus reservoirs during HIV-1infection: previous work has described organelles within mac-rophages as preferential sites for virus accumulation in mac-rophages (114). Expression analysis revealed high levels of

CD63 and major histocompatibility complex type II (MHC II)and low levels of Lamp 1 in these organelles, resembling thecompartments where MHC II molecules undergo the finalstages of maturation. CD63 is also enriched on the surface ofmultivesicular bodies, and it is presumed that HIV-1 particlesmimic the mechanisms utilized in the production of naturalinternal vesicles (128). Thus it is possible that CD63, like CD81and CD82 (see above), may be involved in viral traffickingand/or association with intracellular vesicles for fusion andrelease.

It has recently been shown that HIV-1 infection is inhibitedby anti-CD63 antibodies, but not by antibodies to CD9 orCD81. This was specific for macrophages and HIV-1 strainswhich use the CCR5 coreceptor, as X4 virus and T-cell infec-tion were unaffected by anti-CD63 antibodies (161). Prelimi-nary data also shows that recombinant tetraspanin-LEL pro-teins can also specifically inhibit macrophage infection but, incontrast to antibody inhibitors, can also inhibit CXCR4-tropicvirus; significantly, the effect is seen with a range of tet-raspanin-LELs (including CD9 and CD81) (S. H. Ho, F. Mar-tin, G. W. Moseley, L. J. Partridge, C. Cheng-Mayer, and P. N.Monk, unpublished data). These experiments indicate a rolefor multiple tetraspanins, probably organized in TEMs, in theinfection of macrophages by HIV. The differences observedbetween the studies using tetraspanin LELs and antibodiesmay be due to a number of factors, as neither approach isphysiological. However, tetraspanin LELs are small proteinsand are not prone to the same artifacts as antibodies such as Fcreceptor coengagement, known to suppress HIV replication inmacrophages (119). Alternatively, the differing efficacy of an-tibodies may relate to epitope-specific events, as described foranti-CD82 in syncytium formation (57). It is also possible thatanti-CD63 antibodies bind directly to the viral envelope,known to contain higher levels of CD63 than other tetraspan-ins, thus inhibiting infection by steric hindrance (117). How-ever, the apparent involvement of CD9 and CD81 in HIV-1infection implies that TEMs are involved in HIV-1 infectionand that this role may involve vacuolar targeting and mem-brane fusion events in viral release.

FIV. Feline immunodeficiency virus (FIV) infection also ap-pears to involve tetraspanins, in this case CD9 (164). In 104-C1PBMC cells transfected to express feline (f)CD9, the anti-CD9antibody vpg15 was shown to delay reversibly, but not com-pletely block, reverse transcriptase activity following infection(29). As the inhibitory effect was enhanced upon addition ofantibody postinfection, it appeared that CD9 can affect eventsafter cellular entry. Treatment of chronically infected cells withvpg15 reduced the numbers of budding particles at the plasmamembrane and inhibited FIV spread in cell culture, so it ap-pears likely that viral assembly and/or release (29) is inhibitedby anti-CD9 antibody, indicating a role for fCD9 similar to thatof other tetraspanins in viral infections. FIV can infect CD9-negative 3201 cells (55), implying that CD9 is not essential forFIV infection or at least that other tetraspanins can take overthis role. However, ectopic expression of CD9 in 3201 cellsenhances FIV infectivity, suggesting that CD9 plays a directrole in infection rather than an indirect role via cell signalingpathways affected by anti-CD9 antibody ligation (163).

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PARAMYXOVIRUS

Canine distemper virus (CDV) (a morbillivirus of theParamyxoviridae family) is a negative-stranded RNA virus, sim-ilar to human measles virus, for which the host cell surfacereceptor(s) are currently not identified (46). As for FIV, ananti-CD9 antibody has been shown to inhibit viral infection(83) and transfection of CD9 into cell lines increases viralproduction, leading to a greater number of infectious centersand larger syncytia, consistent with the effects of CD9 expres-sion on syncytium formation by a rhabdomyosarcoma cell line(155). CD9 does not appear to be a receptor for CDV (83), andanti-CD9 antibody does not block CDV attachment to cells(141). The involvement in subsequent events (83), includingmembrane fusion/syncytium formation, is comparable to thatof other tetraspanins in human viral diseases and CD9 in FIV.

TETRASPANINS AS POTENTIAL THERAPEUTICTARGETS FOR VIRUSES THAT INFECT HUMANS

HCV infects 170 million people worldwide, causing liverdiseases that include hepatitis, cirrhosis, hepatocellular carci-noma, and disorders relating to immune dysregulation (125,157). HIV/AIDS is responsible for more than 25 million deaths

since 1984, with an estimated 37.8 million individuals currentlyinfected (Joint United Nations Program on HIV/AIDS, UN-AIDS, 2004). HTLV-1 presently infects 20 million individualsworldwide (32) and is associated with adult T-cell-leukemia/lymphoma and myelopathy/tropical spatic paraparesis. Treat-ments for these viruses include antiviral drugs such as inter-ferons, viral enzyme inhibitors, and viral-cell receptorinteraction inhibitors (24, 25, 35, 62, 71, 100, 157), which cansubstantially lower the viral load (115) but cannot eliminate thevirus. Further problems associated with such treatments in-clude toxicity and side effects, high cost, development of viralresistance, and low efficacy owing to viral genotype specificity/patient variables (11, 43, 61, 101, 108, 121, 145, 157, 171).Efforts to develop conventional vaccines for HIV have beenhampered by potential pathogenicity and poor capacity to elicitprotective immune responses (6, 54, 80, 162). Several new vacci-nation approaches for HIV (e.g., DNA and viral vectors) are inclinical trials (3, 4, 7, 23, 82, 105, 133, 146). However, in commonwith conventional vaccines, most of these strategies have beenshown to be ineffective due either to their poor capacity to elicitimmune protection (40, 85) or to the various mechanisms of HIVimmune evasion, such as high rate of mutations and host genomeinsertion (34, 53, 72, 73, 106, 116, 127).

FIG. 3. Model for the role of TEMs in viral infection. Current evidence (represented in inset) indicates that viral proteins may associate withTEMs by forming direct primary interactions with a tetraspanin (black arrows) which, via secondary/tertiary interactions of tetraspanins (greyarrow), incorporate the viral protein into the tetraspanin web, permitting indirect interaction with tetraspanin-associated molecules (dotted arrow).Various data indicate that, through these interactions, TEMs may be involved in initial viral attachment (no. 1) and entry (no. 2) into the cell. Somedata also indicate that viral interaction with TEMs may couple to intracellular signaling pathways, priming cells for infection or altering immunecell functions (no. 3). TEMs may interact with new viral proteins after infection/transcription in the biosynthesis/secretion pathway (no. 4). Multiplelines of evidence also indicate that TEM involvement in membrane fusion events may be exploited for viral exit from cells whether via direct releaseby secretory vesicles or in exosomes (no. 5 and 6) or via cell-cell fusion to form syncytia (no. 7). In the latter, tetraspanin-associated cell adhesionmolecules (e.g., ICAM, integrins) are likely to mediate intercellular interactions in the fusion process.

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As described above, various groups have identified tet-raspanins as the targets of antibodies which inhibit the infec-tivity of a range of viruses but have generally identified aspecific tetraspanin in each of these studies. However, it ap-pears that at least two tetraspanins may be involved in HTLV-1infection and at least four in HIV-1 infection. Thus, we hy-pothesize that (i) several tetraspanins will be involved in someor all of the viral life cycles here mentioned, and (ii) thattetraspanins are likely to be involved in many other viral in-fections as yet undiscovered. While definition of the preciseroles of TEMs in viral infection awaits further research, it hasalready been shown that peptides based on tetraspanin-LELscan inhibit tetraspanin functions such as sperm/oocyte fusion(51). A peptide inhibitor of only 14 residues, designed to mimicthe region 176 to 189 of CD81 has been shown to inhibit theCD81-E2 interaction (30). With a greater understanding of theassociation of TEMs with viral proteins, particularly the pri-mary ligand interactions, it should become possible to selec-tively target TEMs for therapy. The E2 binding site on CD81has been mapped to specific amino acids in the LEL subloop(31, 160). Based on these findings, several bi-imidazole-basedcompounds that can also inhibit E2 binding to CD81 have beenidentified (160). These resemble the solvent-exposed face ofthe helix of CD81 LEL that contains Phe186, thus mimickingthe E2 binding site on CD81. Two of the compounds have 50%inhibitory concentration values below 100 �M and representan important proof of the concept that tetraspanin-based drugscan disrupt biological function. Thus, targeting TEMs mayprovide a novel strategy to inhibit critical processes of viralinfection.

CONCLUDING REMARKS

The precise nature of the relationship of tetraspanins withviruses is presently poorly defined and is complicated by theapparently disparate functions of specific tetraspanins in infec-tions by different viruses (summarized in Fig. 3). However,much of the data describing this relationship has been derivedfrom in vitro studies. Thus, it is unclear whether modulation ofimmune cell line function by LEL-CD81 ligation, for example,has any genuine significance in vivo. Similarly, while CD81 isknown to interact with HCV coat proteins in vitro, the stage ofinfection at which this interaction occurs in vivo is unknownand need not necessarily occur during cell entry. Indeed theinteraction of CD82 with HTLV-1 proteins occurs postinfec-tion, while CD9 and CD63 appear to affect FIV/CDV andHIV-1 late in infection. Of particular significance is the recentdemonstration that soluble LEL proteins from several differenttetraspanins can inhibit HIV-1 infection and the overlappingeffects of certain antitetraspanin antibodies in syncytium for-mation by rhabdomyosarcoma cells. This suggests that there isfunctional overlap between different tetraspanins involved inviral infection processes, which implies the involvement ofTEMs.

Thus, rather than being passive targets for opportunisticviral attachment to cells, tetraspanins are likely to play a morefundamental role in viral infectivity through the involvement ofTEMs in viral protein maturation/trafficking, in cell-cell fusionevents or in other membrane fusion events involved in viralrelease, processes which have clear parallels in normal TEM

physiology. In this context, the selective primary interaction ofviral protein with a specific tetraspanin (e.g., CD81 with HCVE2) to connect it via secondary/tertiary interactions to tet-raspanin complexes involved in these processes would be typ-ical of the incorporation of nontetraspanin proteins into theTEMs (Fig. 3). Targeting TEMs for viral therapies may there-fore target a principal mechanism in the infectious cycles ofHIV-1, HTLV-1, and HCV which viral mutation would notreadily circumvent, providing new antiviral therapies so ur-gently required by an ever-increasing percentage of the world’spopulation.

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