Virus yoga: the role of flexibility in virus host cell recognition

Virus yoga: the role of flexibility invirus host cell recognitionEugene Wu and Glen R. Nemerow

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, California 92037, USA

Viral capsid proteins and their receptors interact in the

context of the virus particle and the plasma membrane

to initiate virus entry. For the non-enveloped adeno-

virus, flexibility in its receptor-binding protein permits

receptor binding to occur while avoiding steric con-

straints that are imposed by the large virus capsids and

the cell surface and might also facilitate interaction

with secondary receptors. Such flexibility in capsid pro-

tein structure is shared by the structurally related reo-

virus. The presence of hinges in the virus attachment

molecule suggests that proper orientation of the

receptor-binding site for viral adhesion depends on the

flexibility of the receptor or viral ligand, especially for

viruses that require the use of multiple receptors.

As the first step in viral entry, the attachment of virions tothe cell surface via a specific cell receptor is a keydeterminant of viral tropism. Viral adhesion triggers aseries of events that lead to the entry of the viral genomeinto the cell for transcription or translation. For someviruses, viral adhesion triggers the engagement of acoreceptor, which promotes the internalization of thevirus genome. The use of distinct receptors means thatthe virus must display multiple binding sites for thesereceptors. In addition, the cell surface glycoproteinsthat serve as receptors are not usually located at thesame distance from the cell surface. Therefore, matchingreceptor-binding sites on the capsid surface to multiplereceptors might require some stretching and bending ofthe virus attachment proteins and their receptors.

Virus receptor-binding proteins are often modular,with a globular receptor-binding domain and a fibrous ortubular stalk domain. The receptor-binding domains ofinfluenza virus and human immunodeficiency virus (HIV)are easily distinguishable from their stalk domains, asthey are separated by proteolytic cleavage. The stalkdomains also serve to fuse the membrane envelopes ofthese viruses to permit the virus genomes to be depositedinto the cytoplasm.

Sequence and structural analyses of the fibers of non-enveloped viruses, such as adenovirus and reovirus, haverevealed globular head domains that are connected to thecapsid by a long, thin, fibrous domain. These fibrous tailsproject the head domain over 100 A (Angstroms) awayfrom the virus capsid. Emerging evidence indicates thatthe receptor-binding activity located on the globular head

domains can be modulated by elements in the bendablefibrous tails. These studies suggest a role for viralflexibility in viral adhesion and entry into the host cell.

Hinges in the Adenovirus fiber shaft

Adenoviruses are non-enveloped dsDNA viruses thatcause infections in mammals and birds. Human adeno-viruses cause infections in the upper respiratory tract, thegastrointestinal tract and the eye. More than 50 serotypesof human adenoviruses have been isolated and classifiedinto six major subgroups designated A–F. Subgroups B, Cand E are commonly associated with respiratory disease,whereas some subgroup D viruses are causative agentsfor severe and highly contagious eye infections [1]. Theirlarge and genetically easy–to-manipulate genomes, coupledwith their ability to transduce resting and dividing cells,make adenoviruses useful tools for vaccine and genedelivery applications [2].

The adenovirus capsid has been well characterizedstructurally [3,4]. It consists of three main proteins: thehexon, the penton base and the fiber. The hexon is themajor structural protein and constitutes the majority ofthe capsid. At each vertex of the icosahedral adenoviruscapsid is a homopentameric complex of penton baseproteins; the trimeric fiber protein extends from thepenton. The penton base–fiber complex has been isolatedfrom disrupted virions and visualized by negative-stainelectron microscopy. From the side, the penton appearstrapezoidal, whereas the long fiber protein appears to havea thin shaft region and a globular head [5].

Studies on adenovirus entry show that the fiber andpenton base proteins play distinct roles in cell entry [6].The fiber protein initiates entry into the host cell bymediating virus adhesion to the cell surface. The fiberknob binds the cell receptor and tethers the virus to thecell surface. The penton base protein, just below the fiberprotein, binds a coreceptor, which signals for virusinternalization by clathrin-coated pits [7]. For all sub-groups of adenoviruses, except subgroup F [8], thiscoreceptor is an av integrin [6,9]. Ligation to av integrinsactivates multiple signaling molecules that ultimatelypromote actin polymerization [10] and enclosure within anendosome. The virus particle then escapes the endosomeand is transported to the nuclear pore complex. There, theviral DNA is transported into the nucleus, where geneexpression, viral replication and assembly occur [11].

The adenovirus attachment protein, the fiber, protrudesfrom the capsid vertices to bind a cell surface receptor.Corresponding author: Glen R. Nemerow ([email protected]).

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Sequence analyses of the fiber protein found three distinctdomains: an N-terminal tail that interacts with the pentonbase, a central shaft domain of variable length amongsubgroups, and the C-terminal globular knob domain. Thefiber knob of all adenovirus subgroups, except for those ofsubgroup B [12], bind to a 46 kilodalton (kD) membraneprotein called the coxsackievirus and adenovirus receptor(CAR). The CAR-binding site has been located to theloop between the first and second b-strands (AB loop) ofthe fiber knob by structural and mutagenic analyses(Figure 1a) [13,14]. This loop faces the sides of the trimericfiber knob and is close to the shaft domain. CAR has beenshown to mediate cell binding of adenovirus type 2 (Ad2),Ad5 (subgroup C) [15], Ad4 (E), Ad12 (A) and Ad41 (F; longfiber) [12].

The AB loops of the subgroup D members Ad9 and Ad37are very similar to that of CAR-using Ad2 and Ad12(Figure 1b), suggesting that the knob binds CAR in theseviruses. CAR has been observed to bind the Ad9 and Ad37fiber knob but surprisingly fails to mediate viral binding orinfection [12,16,17]. This presents a conundrum: if CAR isrecognized by subgroup D fiber knobs, then why does it notmediate virus-binding to cells? One possibility is that thefiber knob domain is not the only region of the fiber thatregulates receptor interactions.

Recent studies have shown that the fiber central shaftdomain modulates adenovirus adhesion to the cell surfacevia CAR. The fiber shaft domains of different adenovirustypes consist of a variable number of repeats of 15–20amino acids. Sequence analysis indicates that thiscentral domain consists entirely of a b-sheet secondarystructure [18]. Each repeat contains two b-strands con-nected by a short linker containing either a glycine orproline (Figure 2a). Determination of the crystal struc-ture of the Ad2 fiber knob with four b-repeats of the shaftrevealed a novel structural fold termed the triple b-spiral[19], which is characterized by the intertwining ofthree polypeptide chains, similar to a spiral staircase inwhich each step is defined by a b-repeat (Figure 2b). Each

Figure 1. The coxsackievirus–adenovirus receptor (CAR) binds the side of the fiber

knob. (a) Three monomers of the first immunoglobulin-like domain of CAR (yellow

strands) bind a single fiber knob trimer (blue space-filling model) [13]. The black

arrow points away from the virus capsid. The loop between the A and B strands

(AB loop; red) of the knob makes up over 50% of the receptor contact surface. (b)

Knobs of all subgroups, except subgroup B (red), bind CAR and contain similar

sequences in the AB loop. Fibers of subgroup D adenoviruses, such as Ad9 and

Ad37, contain similar sequences in the CAR-contact residues (*) to CAR-binding

fibers of Ad2, Ad5 and Ad12.

* ** *

A Ad12 TPDPPPNCSLIQE

C Ad2 TPDPSPNCRIHSD

C Ad5 TPAPSPNCRLNAE

D Ad9 TPDTSPNCKIDQD

D Ad37 TPDTSPNCTIAQD

E Ad4 TPDPSPNCQILAE

F Ad41 TADPSPNATFYES

B Ad3 GPKPEANCIIEYG

(a)

CARBinders

(b)

TRENDS in Microbiology

Figure 2. The b-repeat of the adenovirus fiber shaft. (a) The b-repeat structure of the adenovirus shaft is characterized by two b-strands (blue strands) connected by a short

b-turn (sticks) [19]. A characteristic b-turn hydrogen bond (green) is formed between the backbone atoms of G362 and G364 of the Ad2 fiber. The third fiber repeat of most

adenovirus serotypes contain a four amino acid insertion in the b-turn (arrow). (b) Repeating units of the b-repeat form a shaft reminiscent of three intertwined spiral

staircases.


(a) (b)InsertionS363G364

G362I365

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b-spiral repeat increases the length of the fiber by ,13 A[19], and the number of repeats correlates with fiber length[20]. The variability of the number of b-repeats in the fibershaft suggests that the central domain might have anadditional biological function.

An interesting correlation can be drawn between thelength of the fiber protein of human adenoviruses andtheir ability to use CAR as a receptor at the cell surface.Subgroup B and D adenoviruses, with relatively shortfibers, do not use CAR, but adenoviruses with longer fibers(11.5 b-repeats or more) do. The ability of subgroup D fiberknobs to bind CAR but not use it to mediate virus entryprompted studies into the importance of the length of thefiber shaft in virus attachment to the cell. Shayakhmetovand Lieber [21] constructed chimeric adenovirus fiberproteins combining the knob of CAR-binding Ad5 withthe short shaft domain of subgroup D Ad9. An adeno-virus vector equipped with this chimeric fiber showed a40–50% decrease in virus attachment and internalizationin CAR-expressing cells compared with a vector that hadthe wild-type Ad5 fiber. A similar study using the shortfiber shaft (11.5 b-repeats) showed a negative impact onadenovirus gene delivery into cells [22], suggesting thatthe shaft can modulate knob–receptor interactions.

However, length is not the only feature of the fiber shaftthat can influence receptor binding. Electron microscopystudies of adenovirus fibers revealed a bend in the fibershafts and also in some instances at the interfaces betweenthe shaft and knob [5,20]. Bending of the fiber at theinterface between the shaft and the knob was attributed toa ‘linker’ sequence between them [19]. Analysis of thecrystal structure of the human Ad2 fiber was unable toclearly define electron density for the linker region(residues 394–396), suggesting that this ‘neck’ region isflexible [19]. In addition, the threefold axes of the knob andshaft deviate by ,28 in the crystal structure [19], andnegative-stain electron microscopy images of the Ad2 fibershowed larger deviations [20]. Bending in the shaft hasbeen correlated with the third b-repeat in fibers of manyhuman adenoviruses. This repeat contains a four aminoacid insertion into the short linker between b-strands ofthe b-repeat [20] (Table 1, Figure 2a). The linker betweenthe two b-strands forms a short b-turn to bring togetherthe anti-parallel strands and faces the hydrophobic core ofthe shaft (Figure 2b). The regular triple b-spiral structureis unlikely to accommodate a four amino acid insertion into

the b-turn, and, as a consequence, is probably partiallyunfolded by the aberrant repeat sequence. The disappear-ance of fiber electron density past 60 A above the penton inthe cryoelectron microscopy structure of Ad2 probablyresults from the bending of the fiber out of the fivefoldsymmetry axes of the icosahedral reconstruction [23]. Theproximity of the bend to the penton strongly suggests thatbending occurs in the third b-repeat.

If the insertion of four amino acids into the b-turn ofthe third b-repeat is responsible for fiber bending, a fiberwithout the same insertion should have a straight shaft.Chroboczek et al. [20] noted that subgroup D adenoviruseshave a normal third b-repeat (Table 1). The cryoelectronmicroscopy structure of Ad37, a subgroup D adenovirus,showed that its fiber is rigid and does not bend at the thirdb-repeat [23]. In contrast to the Ad2 electron microscopystructure, the entire Ad37 fiber could be imaged, includingthe fiber knob, strongly pointing to the third b-repeat as asource of fiber flexibility.

The apparent differences in fiber structures promptedus to explore the possibility that the failure of Ad37 to useCAR was due to its short, rigid architecture. We hypoth-esized that the rigidity of the Ad37 fiber protein (and othersubgroup D adenoviruses) prevented the proper orien-tation of the CAR-binding site on subgroup D adenovirusfiber knobs to facilitate binding. To test this hypothesis weattempted to restore the CAR-binding activity of the Ad37fiber knob by fusing it to the shaft of the long and flexibleAd5 fiber. This fusion resulted in a marked increase invirus binding and infectivity compared with the short andrigid wild-type Ad37 fiber [22]. However, this did notdistinguish between the contributions of length andflexibility. If the flexibility of subgroup C fibers contributedto CAR binding at the cell surface, then making the fiberrigid should negatively influence virus binding andinfection. To test this, the flexible third b-repeat of theAd5 fiber was replaced with the normal third repeat of theAd37 fiber. In contrast to the reconstruction of AD2 andAd12 virus [24], electron density from this chimeric fibercould be observed to nearly 360 A, which corresponded tothe length of the entire Ad5 fiber using cryoelectronmicroscopy [22] and demonstrated that this fiber is morerigid than the wild-type Ad5 fiber. Making the fiber rigidalso drastically reduced cell binding and infection [22],showing the importance of fiber flexibility in virusadhesion. The importance of bending the knob at the

Table 1. Number and alignment of b-repeats of adenovirus fiber proteins from subgroups A–Fa

Subgroup Shaft repeats Representative serotype Third repeat sequence

abcdefgh----ijklmno b

Flexible? Flexibility Refs

A 22.5 12 GS LTAS N NINVLEP L TNTS Yes [24]

B1 5.5 3 GS LEEN I KVN--TP L TKSN Noc [34]

B2 5.5 35 GT LQEN I RAT--AP I TKNN Noc c

C 21.5 2 GN LTSQ N VTTVSPP L KKTK Yes [24]

D 7.5 37 GS LTVN P K--- -AP L QVNT No [23]

E 11.5 4 GK LIAN T VNKAIAP L SFSN Yes?

F (long)d 21.5 41 GD LSSD A SVEVSAP I TKTN Yes?

aThe four amino acid insertions in the b-turn are colored red.bGreen amino acid positions are typically hydrophobic and the blue position (j) is invariably proline or glycine.cCryoelectron microscopy reconstructions of Ad3 penton dodecahedrons [34] and Ad35 virus particles (G.R. Nemerow et al., unpublished) were both able to image the fiber

knob, suggesting that two residue insertions in the third repeat are not sufficient for fiber flexibility.dSubgroup F adenoviruses contain a long and short fiber.




neck has not been explored, although inhibition of neck-bending might also reduce CAR interaction. Studies intothe effects of alterations of the fiber shaft on adenovirusbinding or infection indicate that long, flexible fibers canbind CAR at the cell surface, whereas rigid fibers or fiberswith eight or less repeats cannot (Table 2).

The elucidation of the role of flexibility in the adeno-virus fiber has led us to generate a molecular model ofvirus adhesion to the cell surface. A major differencebetween binding of fiber knob to immobilized CAR andadenovirus adhesion to CAR at the cell surface is theorientation of CAR. CAR is probably randomly oriented onthe solid support, but as an integral membrane proteinCAR is restricted in its orientation at the cell surface.Based on the sequence and structural homology of CARwith junctional adhesion molecules (JAMs) [25], we haveoriented CAR with respect to the cell surface [22]. Therestriction in orientation with respect to the cell surface,compounded with the large size of the adenovirus capsid,precludes adenoviruses with rigid fibers (i.e. subgroup Dadenoviruses) from binding to CAR at the cell surface [22](Figure 3a). Instead of binding CAR, subgroup D adeno-viruses must bind an alternative primary receptor [16,17]and then engage av integrins for internalization [8,23].Binding of a receptor that lies close to the cell surface bythe short Ad37 fiber (,150 A) might make subsequent

interaction with av integrins possible [23]. Subgroup A, C,E and F adenoviruses overcome this impediment bybending their fiber proteins at the third b-repeat and theneck of the shaft (Figure 3b). Bending at two or moreplaces ensures that, once bound to CAR, the capsid is stillfree to adopt many orientations and distances relative tothe cell surface. The capsid fivefold symmetry axis canthen point perpendicular to the cell surface and facilitatepenton base interaction with av integrins (Figure 3b).Such a conformation could cluster integrins and lead tosignaling and virus internalization [24]. Thus, flexibility ofthe fiber shaft could promote both primary and secondaryreceptor-binding.

Bending and stretching in reovirus–receptor

interactions

Reovirus, an RNA virus with a distinct evolutionaryorigin, has remarkable structural similarities to adeno-virus (Box 1). Similar to the adenovirus capsid, the non-enveloped reovirus capsid is ,850 A in diameter. Reovirusattaches to cells via the interaction between fiber-like s1and the cell receptor, the junctional adhesion molecule 1(JAM1). Following attachment, the virus is internalizedinto an endosome. Acidification of the endosome triggersrearrangements of the virus outer capsid and the loss ofsome outer capsid proteins. This intermediate subviralparticle (ISVP) penetrates the endosome and depositsthe viral core into the cytoplasm [26]. Up to this point, theentry mechanism of reovirus is similar to that of adeno-virus. In contrast to adenovirus, however, reovirus repli-cates its dsRNA genome in the cytosol.

Reovirus and adenovirus not only share similar entrypathways, but they also recognize the membrane-distaldomain of homologous receptors (CAR and JAM). Bothreceptors are members of the immunoglobulin (Ig) super-family, with two Ig domain membrane proteins thatdimerize in very similar fashions [22,25]. The G, F, Cand C0 b-strands form the homodimerization interfacefor the membrane-distal Ig domains of both CAR andJAM1. At the centers of both dimer interfaces lie tworesidues of opposite charge that form a pair of salt bridges(K123-D54 of CAR and R59-E61 of human JAM). Theseprimary, tertiary and quaternary structural studies revealstriking similarity between adenovirus and reovirusreceptors (Figure 4a).

Despite having little sequence similarity with theadenovirus fiber, the 40–50 nm reovirus s1 protein has

Table 2. Fiber shaft alterations and their effects on adenovirus binding or infection of CAR-expressing cells

Knob Shaft b-repeats Flexible? Cell binding and infectiona Refs

Ad5 Ad5 (wild-type) 21.5 Yes þ

Ad5 Ad9 7.5 No 2 [21]

Ad9 Ad5 21.5 Yes þ /2b [21]

Ad5 Ad5 þ Ad2 Up to 32 Yes 2c [35]

Ad5 Ad37 7.5 No 2 [22]

Ad37 Ad5 21.5 Yes þ [22]

Ad5 Ad5 (1–3, 18–21.5) 7.5 Yes 2 [22]

Ad5 Ad5 (1–2, 4–20); Ad37 (3, 7) 21.5 No 2 [22]

Ad5 Ad5 (1–2, 17–21.5) 7.5 Yes 2 [36]

aVirus binding or infection of CAR-expressing cells.bLow binding to cells, but higher than wild-type Ad9 fiber.cLow infection, possibly due to inability to bind av integrins; cell-binding not measured.

Figure 3. Models of adenovirus interaction with receptors at the cell surface. (a)

Ad37 is unable to overcome steric hindrance presented by the capsid and the cell

surface because the rigid Ad37 fiber (green) cannot bend to correctly orient the

knob. (b) Ad5 overcomes the steric hindrance by bending the flexible fiber protein.

The adenovirus fiber contains two hinges (curved arrows, background particle),

which allow the simultaneous binding of CAR (magenta) by the fiber and multiple

av integrins (red) by the penton base (yellow). The domain arrangement of CAR at

the cell surface was modeled using the crystal structure of the junctional adhesion

molecule [31].


(a) (b)




an unexpectedly similar architecture to that of adenovirus[27]. The tail domain of the s1 protein consists of a coiled-coil region and a region with b-repeats of 15–20 aminoacids [28]. Sequence analysis of s1 proteins from threeserotypes of reoviruses indicates that, similar to theCAR-binding site on the adenovirus fiber knob, theJAM-binding site lies along the side of the s1 head [29].Also, cryoelectron microscopy image reconstructions ofreovirus show that the s1 protein electron density becomesdiffuse or discontinuous 46 A past the l2 pentamer. Theincomplete s1 reconstruction was attributed to s1 bending[30]. Crystallographic analysis of the s1 head with severalb-repeats revealed a striking similarity to the three

dimensional structure of the adenovirus fiber, despite nodiscernable sequence homology [29]. Similar to the fiber,s1 forms a homotrimer with a triple b-spiral tail leading toan eight-stranded b-barrel head. Of particular interest,the crystallized s1 protein contained a b-repeat with a fouramino acid insertion between the b-strands of the repeat.This repeat was completely without secondary structureand appeared unfolded. The lack of structure resulted in alarge bend (,238) at the second to last repeat in thereovirus tail. This structural evidence strongly supportsthe notion that the four amino acid insertion into thethird b-repeat of the adenovirus fiber shaft disrupts tripleb-spiral structure and allows the fiber to bend (Figure 4b).

Box 1. Similarities in adenovirus and reovirus entry

Adenovirus and reovirus, two non-enveloped viruses, are vastly

different in genetic and capsid organization. Adenovirus contains a

linear double-stranded DNA genome, whereas reovirus contains a

segmented double-stranded RNA genome. In contrast to the single-

layered capsid of adenovirus, the reovirus virion has two layers: a core

and an outer capsid. There is no known sequence homology between

the two viruses.

Despite these differences, the two viruses share entry mechanisms.

The large capsids (850–900 A in diameter) of adenovirus (Figure Ia) and

reovirus (Figure Ib) bind to primary receptors via fibrous protrusions

from the capsid vertices (Figure Ic). The adenovirus penton binds to a

secondary receptor (av integrins) to induce internalization into an

endosome. A secondary receptor has not been identified for reovirus.

Endosome acidification leads to dissociation of capsid components and

structural rearrangements for both viruses. These structural changes

lead to virus escape into the cytoplasm.

Once in the cytoplasm, the two viruses use different mechanisms for

gene expression. For reovirus, any outer capsid components are shed

and the viral core remains intact in the cytoplasm. The reovirus core

carries its own transcription machinery and extrudes viral mRNAs for

translation. By contrast, adenovirus must transport its DNA genome

into the nucleus. Upon escape from the endosome, the partially

dissociated adenovirus capsid is transported via microtubules to the

nucleus. There, the viral genome is released and imported into the host

cell nucleus, where it uses the host cell transcription machinery for gene

expression. For a review on adenovirus and reovirus entry processes,

see Ref. [37].

Figure I. Comparison of adenovirus and reovirus structure and entry. (a) The

adenovirus capsid has pseudo T ¼ 25 symmetry and is approximately 900 A

in diameter. Fiber proteins protude from the capsid vertices. (b) Reovirus con-

tains two capsid layers, a T ¼ 1 core (purple and yellow lines) and a T ¼ 13

outer capsid (blue and green lines). The reovirus capsid is approximately

850 A in diameter. s1 proteins protrude from the capsid vertices. (c) Adeno-

virus (blue) and reovirus (purple) use similar entry mechanisms. Both viruses

attach to cells via an integral membrane protein with two immunoglobulin-

like domains (green). Adenovirus also binds av integrins (red) as a secondary

receptor. Adhesion to the cell leads to receptor-mediated endocytosis. Both

viruses partially disassemble and escape intracellular vesicles. Partially disas-

sembled adenovirus is transported to the nucleus via microtubules, and its

genome is probably transported into the nucleus through the nuclear pore.

Reovirus genes are transcribed from the reovirus core particle directly in the

host cell cytoplasm.


H+ H+

Nucleusproteases

core

(a)

(c)

(b)

Adenovirus Reovirus

microtubule




Based on the bent crystal structure, Chappell andcolleagues [29] also proposed that flexibility at the neckof s1 could be required for interaction with JAM.

In addition, negative-stain electron micrographs ofreovirus s1 also reveal a second hinge near the center ofthe tail [27]. The location of the second hinge has not beenassociated with any particular sequence in the s1 shaft.Interestingly, s1 density extends to 110 A in ISVPs (versus46 A on virions), suggesting that s1 is less flexible onISVPs than on virions. The modulation of s1 flexibilitymight play an as yet unknown role in virus internalizationor endosome penetration. The presence of two hinges inthe reovirus s1 suggests that reovirus must also properlydistance and orient its capsid with respect to the cellsurface for interaction with a secondary receptor, althoughthe identity of this secondary receptor is not known.

Perspectives

Non-enveloped virus particles are often represented assimple static spheres or icosahedral particles outside thecell. For adenovirus and reovirus, however, this might notbe the case. These viruses use a distinct attachmentprotein that is displayed outside the capsid by a tether.These ligands do not act independently, however, as theirelongated structures must interact with high affinity to areceptor at the cell surface. The capsid and the cell surfacepresent large three-dimensional obstacles to overcome forthe adenovirus fiber knob and reovirus s1 head to attach totheir receptors. We believe these viruses overcome large

steric hindrances by using bendable fibers, thus allowingthe head domain to adopt multiple conformations relativeto the receptor and the cell surface.

Our model has direct implications for virus–receptorinteractions for the rigid fibers of subgroup D adeno-viruses, such as Ad37. Without a flexible fiber, subgroup Dadenoviruses must rely on binding a flexible receptorprotein or a receptor protein already pre-positioned forvirus attachment. Arnberg and colleagues [16] havereported that Ad37 binds sialic acid located on an unknownglycoprotein. Wu and colleagues [17] have characterizedAd37-binding to 50 and 60 kD membrane proteins. Until thereceptors for Ad37 and other subgroup D adenoviruses areidentified, it is unclear how the rigid fibers of subgroup Dadenoviruses will overcome the steric hindrance presentedby the cell surface and the large virus capsid.

This model of non-enveloped virus–cell interactionrelies on several assumptions. First, we have assumedthat the cell membrane is essentially a planar surface asviewed by the virus particle. For a virus particle less than100 nm in diameter, a spherical cell ,100 times itsdiameter would, in general, appear flat when the virus isvery near the cell surface. The curvature and local defor-mations of the cell membrane would probably not besufficient to accommodate the large (308 or more) overlapbetween the cell surface and the Ad37 capsid when boundto CAR. We also assume that the receptors (CAR and JAM)are rigid molecules with respect to the cell surface.These type I membrane proteins have a single a-helix

Figure 4. Similarities between the adenovirus and reovirus receptors and ligands. (a) CAR (blue) and the junctional adhesion molecule (JAM; green) are both immunoglobu-

lin (Ig) superfamily members that dimerize in nearly identical orientations [25], as viewed from the top and side. Residues of opposite charge (K123 and D54 of CAR and

R59 and E61 of JAM; sticks) face each other near the twofold symmetry axes and constitute a central portion of the GFCC0 interface. The second Ig domain of CAR (blue

ovals) has not been structurally characterized. (b) The JAM-binding reovirus ligand, s1, is structurally homologous to the adenovirus fiber [29]. Four amino acids (sticks)

are inserted into the head-proximal b-repeat, resulting in an unfolded bend and a large deviation between the threefold symmetry axes of the shaft (blue triangle) and the

head (red triangle). The putative JAM-binding site, similar to the CAR-binding site of the adenovirus fiber, is also located on the side of the s1 head (arrow) [29].


(a) (b)

JAM?

Bend

D54-K123

E61-R59

K123-D54

R59-E61

??




transmembrane domain, which probably confers rota-tional freedom, but does not change the angle of thedomains relative to the cell surface. The linkers betweenthe Ig domains are quite short and, in the cases of murineand human JAM crystal structures, are not flexible. Thelinkers between the second Ig domain and the transmem-brane domains are also quite short, but no structuralstudies have described these regions.

A better understanding of the importance of flexibilityin viral adhesion might facilitate the design of antiviralsthat block viral adhesion to the cell surface by inhibitingthe bending of virus fibers or their receptors. Smallmolecules or antibodies that bind to hinges in viral fibersor their receptors could limit the orientations andconformations of these molecules and thereby modulatethe virus–receptor interaction. In a multi-receptor inter-action, such as that for adenovirus, one might envisiontrapping the fiber in a rigid conformation to interruptadhesion or in a conformation that is too extreme tofacilitate penton interaction with the secondary receptor(av integrins) and inhibit virus internalization. In the caseof the interaction between HIV and its receptor, theimmunoregulatory molecule CD4, it has been found thatthe receptor contains hinges and bends at the linkersbetween the second and third Ig domains [31] and betweenthe fourth Ig domain and the transmembrane region [32].Alterations to these putative hinge regions of CD4 bymutagenesis [32] or antibody-binding [33] allow the virusto bind the first Ig domain (D1) of CD4, but negativelyaffect membrane fusion. These studies indicate that afterHIV gp120 binds the membrane-distal D1 of CD4, thevirus must be brought closer to the cell surface by thebending of CD4 to interact with the multipass trans-membrane chemokine receptors for membrane fusionand deposition of the viral nucleocapsid into the cyto-plasm. Interruption of this process might be an effectiveantiviral strategy.

Conclusions

At the surface, virus–receptor interactions appear to beone of the simplest steps of virus entry. Upon furtherinvestigation, we find that the mere presence of receptor isnot sufficient for virus adhesion to the cell and that theprecise orientation of all molecules involved is importantfor viral entry, particularly for multi-receptor systems,such as adenovirus, reovirus and HIV. To properly orientviral ligands to their respective receptors, these viruseshave either evolved flexible attachment proteins orusurped flexible receptors for entry. Further studies ofthe reoviruss1 protein and CD4 will be required to confirmthe importance of flexibility for these systems. Perhapsfurther work will enable researchers to take advantage ofthese features to improve antiviral and gene therapy.

Acknowledgements

We thank Joan Gausepohl for preparation of the manuscript and LarsPache for comments on the manuscript. We also wish to express ourgratitude to Dr. Phoebe L. Stewart (Vanderbilt University, Nashville, TN)for providing structural data on adenovirus type 35 pseudotyped virus.E. Wu is supported by the LJIS Interdisciplinary Training Program andthe Burroughs Wellcome Fund.

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Virus yoga: the role of flexibility in virus host cell recognition

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