multistep entry of rotavirus into cells: a versaillesque dance

Multistep entry of rotavirus into cells:a Versaillesque danceSusana Lopez and Carlos F. Arias

Departamento de Genetica del Desarrollo y Fisiologıa Molecular, Instituto de Biotecnologıa,

Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos 62210, Mexico

Rotavirus entry into a cell is a complex multistep pro-

cess in which different domains of the rotavirus surface

proteins interact with different cell surface molecules,

which act as attachment and entry receptors. These

recently described molecules include several integrins

and a heat shock protein, which have been found to be

associated with cell membrane lipid microdomains. The

requirement during viral entry for several cell mol-

ecules, which might be required to be present and orga-

nized in a precise fashion, could explain the selective

cell and tissue tropism of these viruses. This review

focuses on recent data describing the virus–receptor

interactions, the role of lipid microdomains in rotavirus

infection and the mechanism of rotavirus cell entry.

The initial events in a viral infection consist of binding ofthe virus to the surface of the host cell, followed bypenetration of the virus particle into the cytoplasm of thecell. The success of these events depends on recognition ofspecific receptors on the cell surface by the virus;consequently, receptors are important determinants ofviral tissue tropism and pathogenesis. The formerlycommon belief that viruses recognize a single cell receptoris becoming more the exception than the rule, and theinteraction of a virus particle with multiple receptorsduring cell entry has become a frequent observation.

Several viruses use at least two different receptors tointeract with their host cells: (i) the binding receptors,which in general allow the virus particle to rapidly attachto the cell surface, and (ii) receptors that are used by thevirus after binding to the cell, which are referred to usingdifferent terminology, such as post-binding, post-attach-ment, entry, fusion, internalization, secondary or co-receptors, depending on the function that they areknown or proposed to play during the process of virusinfection. In this review, we use the general term of post-binding receptor to encompass all cell receptors that areknown to interact with the virus particle after its initialattachment to the cell surface, regardless of the particularstep that they mediate (Table 1). In some instances, suchas in the case of human immunodeficiency virus-1 (HIV-1),coxsackievirus A9, human cytomegalovirus (HCMV) andadenovirus [1–4], the multiple interactions that take placebetween the virus and cell surface molecules have beenproposed to occur in a sequential manner. Theseinteractions frequently induce conformational changes inthe viral surface proteins, which expose hidden proteindomains that are essential for penetration of the virus intothe cellular cytoplasm [5–7]. These domains couldrepresent potential sensitive targets for novel antiviralagents designed to interrupt the entry of these viruses, asit has been shown for HIV-1 [8].

Table 1. Viruses that use more than one receptor to infect the cell and usage of rafts during their life cyclea,b

Virus Binding receptor(s)a Post-binding receptor(s)a Raft-associated function Refs

Adenovirus CAR, SA, HS avb3, avb5 – [4,56]

Epstein–Barr virus CD21 HLA-II, avb3, avb5, a5b1 Entry [57,58]

Coxsackievirus A9 avb3 GRP78, MHC-I Entry [3]

Echovirus types 1 and 11 DAF, HS a2b1, avb3 Entry, traffic [47,59]

Foot and mouth disease virus SA avb1, avb3, avb6, a5b1 – [6,59]

Herpes simplex virus 1 and 2 HS HVEM, nectin 1, 2 Entry, budding [58,60]

Human herpes virus 8 HS a3b1 – [58]

Human immunodeficiency virus 1c CD4, Gal-C CCR5, CXCR4 Entry, budding [1,6,47]

Murine polyomavirus SA a4b1 – [61]

Respiratory syncytial virus HS ICAM1 – [6]

Reovirus SA JAM – [62]

Rotavirusd SA, a2b1 a2b1, avb3, axb2, hsc70, a4b1 Entry, assembly [12,14,21,22,24,25,46,63,64]

aThe classification used here reflects the most widely accepted roles in the literature for the receptors included, although in some cases a given cell molecule has been reported

to be used as both binding and post-binding receptor, depending on the virus strain or the type of cell studied.bAbbreviations: CAR, Coxsackie adenovirus receptor; DAF, decay accelerating factor; GRP78, glucose regulated protein 78; Gal-C galactosyl-ceramide; HLA, human leukocyte

antigen; HS, heparan sulfate; HVEM, herpes virus entry mediator; ICAM, intracellular adhesion molecule; JAM, junction adhesion molecule; MHC I, major histocompatibility

antigen class I; SA, sialic acid.cThere are more cellular molecules that have been proposed to function as binding and post-binding receptors for HIV-1, depending on the cellular type characterized; listed

here are the best characterized receptors.dIntegrin a2b1 has been described as both a binding and post-binding receptor.

Corresponding author: Carlos F. Arias ([email protected]).

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Rotaviruses are large non-enveloped viruses and, todate, five distinct interactions between cell receptors anddifferent domains of the virus surface proteins have beendescribed. Some of these interactions probably occur in asequential and coordinated manner, from the initialcontact of the virus with the cell surface to the penetrationof the virus particle into the cytoplasm of the cell. Thiscomplex and elegant multistep process of viral entry isreminiscent of the minuet of the 18th century court ballsheld in Versailles, France; an extremely elegant andgraceful dance where the couples started at one end of aline of contra-dancers and moved with small steps throughthe line touching the hands of different partners, sharingfrequent kisses, to finally exit the line of dancers at theother end. In this review we propose to summarize recentfindings on the identification and characterization of theparticipants, both cellular and viral, involved in theVersaillesque dance that rotaviruses perform to enterthe cell. This represents the first phase of the viralreplication cycle, followed by mRNA transcription, proteinsynthesis, early morphogenesis, RNA replication andvirus particle maturation and release [9].

Rotaviruses

Rotaviruses are the leading etiologic agent of severediarrheal disease in infants and young children world-wide, being responsible for an estimated 500 000 deathseach year [10]; therefore, there is an urgent need todevelop effective vaccination and therapeutic strategiesto combat these viruses. Fundamental for these

developments is a thorough basic understanding of themolecular mechanisms that rotaviruses use to interactwith their host cell and replicate.

Rotavirus particles consist of three concentric layers ofprotein. The innermost layer is formed by the proteinknown as VP2, which surrounds the viral genome(composed of 11 segments of double-stranded RNA), andproteins VP1 and VP3; together these proteins constitutethe core of the virus. The addition of VP6 to the VP2 layerproduces double-layered particles. The outermost layer,characteristic of infectious triple-layered particles, iscomposed of two proteins, VP4 and VP7. The smoothsurface of the virus is made up of 260 trimers of VP7, and60 dimers of VP4 extend as spike-like structures from thevirus surface (Figure 1) [9]. VP4 has essential functions inthe virus life cycle, including receptor-binding and cellpenetration. Treatment of the virus with trypsin results inthe specific cleavage of VP4 into the polypeptides denotedas VP8 and VP5 (in some publications these are termedVP8* and VP5*) and confers icosahedral ordering on theVP4 spikes [11], which is essential for the virus to enter thecell. VP7 is a calcium-binding protein that has beenrecently shown to interact with rotavirus cell receptorsafter the initial attachment of the virus to the cell surface[12] (S. Zarate et al., unpublished).

Rotaviruses have a specific cell tropism in vivo,infecting primarily the mature enterocytes of the villi ofthe small intestine, suggesting that these cells containspecific receptors for the virus. However, recent reportssuggest that extra-intestinal spread of the virus takes

Figure 1. Cryo-electron microscopy reconstruction of rotavirus particles. (a) Surface representation of the virus structure at 22 A. The smooth external surface of the virus is

made up of 780 copies of glycoprotein VP7 (in yellow) organized as trimers, and extending ,12 nm from the VP7 surface are 60 spike-like structures, formed by dimers of

VP4 (in red) [9]. (b) The outer VP7 layer was removed from the reconstruction to appreciate the interaction of VP4 with the intermediate VP6 layer (blue). (c) Cartoon

representation of an isolated VP4 spike depicting its location with respect to the VP7 layer (yellow) and VP6 trimers (blue outline). The VP4 spike is formed by two globular

heads attached to a square-shaped body, which is connected to a rod-like domain that merges with a globular base. This large globular domain resides below the VP7 layer

and interacts extensively with VP6 [37]. The sialic acid (SA)-binding domain and the putative fusion domain, located at the distal end of the spikes [35,37], are indicated by

violet and gray circles, respectively. The localization of the VP8 and VP5 domains, as suggested by Tihova et al. [37], is indicated. The vertical pink bar represents the region

where the heat shock protein hsc70-binding domain of the protein is predicted to be located. The images in this figure are a courtesy of Dr. B.V.V. Prasad, Baylor College of

Medicine, Houston, TX, USA.

TRENDS in Microbiology

(a) (b)

VP8

VP5

(c)


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place during infection [13], indicating a wider host-tissuerange than previously thought. In vitro, rotaviruses bindto a wide variety of cell lines, although only a subset ofthese (including cells of renal or intestinal origin, andtransformed cell lines derived from breast, stomach, boneand lung) is efficiently infected [14]. This suggests that thebinding of rotavirus to cells is promiscuous and theinteraction with cell receptors that is responsible forviral entry probably occurs at a post-binding step.

Rotavirus receptors

It is generally accepted that N-acetylneuraminic acid, alsoknown as sialic acid (SA), is required by some animalrotavirus strains to attach to the cell surface. Theinfectivity of these strains is greatly diminished by thetreatment of cells with neuraminidase (NA); consequently,these strains are NA-sensitive. By contrast, manyanimal strains and most strains isolated from humansare NA-resistant [15]; however, this does not mean thatthese strains do not use SA for cell attachment becauseSA moieties that are internal in oligosaccharidestructures are either less sensitive or not sensitive atall to NA [16]. Furthermore, although many rotavirusstrains apparently do not need to bind to SA to infectcells in static culture conditions, this does not implythat binding to these acid sugars is not important forenterocyte infection in the gut within a more dynamicenvironment. The interaction of rotavirus with SA hasbeen shown to depend on the VP4 genotype of the virusand not the species of origin [17]. Ganglioside GM3 hasbeen suggested to serve as the SA-containing receptorfor the porcine rotavirus strain OSU [18], whereasganglioside GM1 (NA-resistant) was described to be thereceptor for the NA-resistant human rotavirus strains KUNand MO [19]. It has also been suggested that rotaviruses ofdifferent origins recognize galactose as a component of theglycoprotein receptors in MA104 cells [20].

Recently, several integrins have been implicated asrotavirus receptors. Rotavirus VP4 contains tripeptidesequence motifs for integrins a2b1 and a4b1, whereas VP7contains integrin ligand sites for integrins axb2 and a4b1[21,22] (Box 1; Figure 2). Antibodies to these integrins, aswell as peptides that mimic their ligand sites, havebeen shown to block the infectivity of NA-resistant andNA-sensitive rotavirus strains [12,14,21–23]. Integrinavb3 has also been shown to be involved in the cellentry of several rotavirus strains at a post-attachmentstep [12,24]. In addition, cell-surface heat shock cognateprotein hsc70 was also implicated as a post-attachmentreceptor for both NA-sensitive and NA-resistant rota-viruses [25]. It has also been recently suggested that somerotavirus strains can infect the cell independently ofintegrins, and it has been shown that the integrin usagecorrelates with the VP4 serotype and is independent of boththe VP7 serotype and the NA-sensitivity of the virus [12].

Virus–receptor interactions

Several lines of evidence suggest that rotaviruses interactsequentially with several cell surface molecules to enterthe cell, using different domains of the virus surfaceproteins VP4 and VP7 during this process [12,21,23–30].

This evidence has been obtained through: (i) a rotaviruscompetition infection assay that detects virus competitionat both binding and post-binding steps [28]; (ii) thecharacterization of a mutant virus that binds to the cellsurface with a modified specificity [30,31]; (iii) the use ofspecific receptor ligands [21,22,24,29]; and (iv) the use ofantibodies to both cell receptors and viral proteins thatblock virus infection by preventing either binding or post-binding events [12,14,21–24,29]. Altogether, this infor-mation has allowed the dissection of the followingsequence of events. The initial contact of a NA-sensitivevirus strain with the cell surface is through a SA-containingcell receptor, most probably a ganglioside, using the VP8domain of VP4. This initial interaction of the virus with SAprobably induces a subtle conformational change on VP4that allows the virus to interact with integrin a2b1 throughVP5. After this second interaction, at least one (and up tothree) additional interaction takes place. These interactionsinvolve theviral surfaceproteinsVP5andVP7, the integrinsavb3 and axb2, and the heat shock protein hsc70 (Figures 2and 3). Whether these last three interactions occursequentially or alternatively has not been established. Theviral and receptor domains involved in each virus–receptorinteraction are described in the following sections.

SA–VP8

NA-sensitive rotavirus strains initially contact SA on thecell surface through the region encompassed between theamino acids located at positions 93 and 208 of the VP8domain of VP4 [32,33], where some residues that are

Box 1. Integrins

Integrins are a family of cell surface receptors that mediate the

interaction between the cell surface and the extracellular matrix

(ECM) and also important cell–cell adhesion events. These inter-

actions play a crucial role in the regulation of cell proliferation,

migration, differentiation and survival. Integrins are transmembrane

heterodimers composed of non-covalently associated a and

b subunits. Both subunits have an extracellular N-terminal domain,

a single transmembrane region and a short cytoplasmic domain.

Human integrins contain at least 18 different a and 8 different

b subunits, which form 24 different heterodimers. Each integrin

heterodimer has distinct ligand-binding specificity and signaling

properties. Integrins can signal through the cell membrane in either

direction: The extracellular binding of integrins to molecules of the

ECM, such as fibronectin, vitronectin, laminin and collagen, is

regulated from the inside of the cell (inside-out signaling). By

contrast, integrin-binding to ECM can relay signals from the outside

of the cell to promote cytoskeletal reorganization and intracellular

signaling (outside-in signaling) [65]. The integrin-recognition motifs

on several integrin ligands have been described (Table I) [66]. Several

viruses and bacteria, which contain integrin-binding motifs on their

surface, take advantage of this family of proteins to gain access into

the cell. In addition, some viruses also interact with integrins through

non-typical sequence motifs [59].

Table I. Some integrin-recognition motifs

Integrin Binding site

avb3, avb5, avb6, a5b1, aIIbb3 RGD

a2b1 DGEA

a4b1 EILDV, IDA

axb2 GPRP

Review TRENDS in Microbiology Vol.12 No.6 June 2004 273



crucial for SA-binding have been identified by alanine-scanning mutagenesis [34] and analysis of the crystalstructure of the central region of VP8 complexed to SA [35].These results have shown that the sialoside-binding site ofVP8 is an open-ended shallow groove (formed by the sidechains of R101, V144, Y155, K187, Y188, Y189 and S190;the one letter amino acid code designation is used) locatedat the tips of the dimeric globular heads of VP4 [36,37]. TheVP8 core has been shown to bind a-anomeric SA with a Kd

of 1.2 mM and does not require additional carbohydratemoieties for binding. In addition, VP8 was shown not todistinguish 30 from 60 sialyllactose and has approxi-mately tenfold lower affinity for N-glycolylneuraminicthan for N-acetylneuraminic acid [36]. The broadspecificity and low affinity of SA-binding by this proteinsupports the suggestion that more specific interactions thatoccurafterSA-bindingareresponsible for rotaviruscell-typeand host specificity [24,28,36].

Integrin a2b1–VP5

In addition to the existence of natural NA-resistantrotavirus strains, mutants that do not need SA to interactwith the cell surface can be isolated from NA-sensitiverotaviruses [31,38]. The characterization of one suchmutant, known as nar3, indicates that it attaches to the

cell surface by interacting with integrin a2b1, whereas theparental NA-sensitive simian rotavirus strain RRV inter-acts with this integrin at a post-attachment step followingits initial binding to a SA-containing molecule. This wasalso shown to be the case for the NA-resistant rotavirusstrain WC3, which interacts with a2b1 at a post-attach-ment level. [14]. By contrast, it has recently been reportedthat integrin a2b1 can be used by NA-resistant (Wa) aswell as NA-sensitive (NCDV, RRV and SA11) rotavirusstrains to bind to the surface of MA104 cells [12],suggesting that a2b1 can function either at a binding orpost-binding stage. In addition, the binding of rotavirusSA11 to K562 cells (non-susceptible to rotavirus infection)transfected with the a2 integrin gene was shown to beenhanced by treatment with a monoclonal antibody to b1that induces the high-affinity state of integrin a2b1 forligand-binding [39], suggesting that the binding ofrotavirus to susceptible cells might be influenced by theactivation state of b1 integrins.

The interaction of both RRV and nar3 rotavirus strainswith integrin a2b1 has been shown to be mediated by theDGE integrin-recognition motif, located at amino acidpositions 308 to 310 of VP4, within the VP5 domain [23].Direct binding of a recombinant VP5 protein to the a2integrin subunit has also been shown recently, and the

Figure 2. Structural features of the rotavirus surface proteins. VP7 is a calcium-binding protein of 326 amino acids that forms a layer on the surface of the viral particle. I and

II denote the two N-terminal hydrophobic regions of the protein. The glycosylation site known to be used by simian rotavirus SA11 at amino acid position 76 is indicated by

three hexagonal shapes. VP7 contains the LDV (at amino acid positions 237 to 239) and GPR (at amino acid positions 253 to 255) tripeptide sequence-binding motifs for

integrins a4b1 and axb2, respectively. A region ranging from amino acid positions 161 to 169 (CNP) interacts with integrin avb3. The virus-neutralizing antibodies directed

to VP7 have been mapped to three major antigenic domains, named A, B and C, which are located at amino acid positions 86 to 101, 142 to152, and 208 to 221, respectively.

VP4 is the spike protein of the virus. In the rhesus rotavirus strain RRV it is 776 amino acids long and contains several discrete functional domains. An arrow indicates the

trypsin cleavage region, where cleavage at arginines 231, 241 and 247 remove a tract of 16 amino acids. The numbers indicate the boundaries between VP8 and VP5. In

VP8, the hemagglutination domain (HA) (amino acid positions 93 to·208) is shown; the asterisks below this domain indicate the amino acids that have been shown to be

important in the sialic acid (SA)-binding activity of this protein (amino acid positions 101, 144, 155, and 187 to 190). The blue arrowheads show the amino acid positions

where the binding of neutralizing monoclonal antibodies has been mapped. The disulfide bridges between C203 and C216, and between C318 and C380, are indicated by

S ¼ S. In VP5, the positions of the DGE and IDA tripeptide sequence-binding motifs for integrins a2b1 and a4b1, located at amino acid positions 308–310 and 538–540,

respectively, are indicated. The hydrophobic region (HR), which has been proposed to be a putative fusion domain (amino acid positions 385–404), and a predicted heptad

repeat (amino acid positions 494–554), which might form part of a coiled-coil structure, are also depicted. The region between amino acids numbered 642–645 contains

peptide KID, known to interact with the heat shock protein hsc70.


HA

VP8

Trypsincleavage

DGE IDA

Coiled-coil

HRS=S

S=S KID

VP5

B CA

100 200 300

CNP

100 200 300 400 500 600 700

326142–152 208–221I II1 86–101

GPRLDV

VP4

VP7

776248

1 231∗∗ ∗∗∗ ∗ ∗




I domain of this integrin was found to be both necessaryand sufficient for this binding [12]; in addition, the DGresidues in the DGE motif of VP5 were found to beessential for this interaction [12].

Hsc70–VP5

After the initial contact with SA and integrin a2b1, thevirus interacts with three additional proteins on the cellsurface: hsc70 and the integrins avb3 and axb2. The viralinteraction with hsc70 is mediated by a domain in VP5 thatis located between amino acid residue positions 642 and659. A synthetic peptide that mimics this VP5 region(peptide KID) and antibodies to hsc70 block the infectivityof rotavirus but not its cell-binding, indicating that VP5interacts with hsc70 at a post-attachment step [29]. Usinga different approach, a similar region (amino acids atpositions 650 to 657) in the VP5 protein of rotavirus strainCRW8 was selected from a VP4 phage display librarybecause of its ability to bind to the surface of MA104 cells[40]. The region represented by peptide KID is notconserved among different rotavirus strains, suggesting

that the hsc70–VP5 interaction is not strictly sequence-specific. It is not known if the chaperone activity of hsc70plays an active role during rotavirus entry, however, it hasbeen recently found that hsc70 interacts with the virusthrough its ligand-binding site, and known ligands of thisprotein efficiently block the infectivity of rotavirus at apost-binding step (J. Perez-Vargas et al., unpublished).

Integrin avb3–VP7

The interaction of rotavirus with integrin avb3 has beenshown to be independent of the RGD motif that is presentin natural ligands of this integrin [24]. Using reassortantviruses that have one surface protein derived from anintegrin-dependent strain and the second surface proteinfrom an integrin-independent virus, it was recently shownthat the interaction of the virus with avb3 correlates withthe presence of a VP7 protein derived from the integrin-using virus [12]. Furthermore, a pairwise alignment usingthe surface protein sequences of rotaviruses andhantaviruses (which also use integrin avb3 in a RGD-independent manner [41]) identified a region containing

Figure 3. A model for the early rotavirus–cell interactions. On the basis of the findings described in this review, we propose a model for the early interactions of rotavirus

with the host cell. This model is illustrated with a neuraminidase (NA)-sensitive virus strain. The initial contact of the virus with the cell surface is through a

sialic acid (SA)-containing cell receptor, most probably a ganglioside, using the VP8 domain of VP4. This initial interaction of the virus with SA probably

induces a subtle conformational change on VP4, which allows the virus to subsequently interact with the I domain of integrin a2b1 (small purple circle on the

a2 integrin subunit) through the DGE domain on VP5. After this second interaction, three more interactions take place, although their order of occurrence has

not been established. These interactions are between: (i) the KID domain of VP5 and the ligand-binding domain of hsc70, (ii) the CNP region of VP7 and

integrin avb3, and (iii) the GRP domain of VP7 and integrin axb2. Based on the topology of VP4 and on the fact that no neutralizing antibodies have been

mapped to the C-terminal region of VP5, the VP4 protein domain represented by peptide KID is probably located at the rod-like domain of the spike, near the

surface of the VP7 layer, in a region of the protein that is not readily accessible. The initial contacts of the virus with the cell surface might trigger a confor-

mational change in VP4 to facilitate the interaction of VP5 with hsc70 and the interaction of VP7 with integrins. With the exception of SA, all other receptor

molecules described appear to be used by most rotavirus strains to infect cells. However, whether the interactions VP5–hsc70, VP7–axb2 and VP7–avb3 are

sequentially or alternatively used by different viruses (i.e. if they are redundant or not) to infect a given cell, or by a given virus strain to infect different cell

types, is not known. Regardless of which receptors are used, the various virus–receptors interactions probably induce conformational changes in the viral proteins,

which ultimately lead to penetration and uncoating of the virus (removal of the surface proteins from the infectious virus to yield the transcriptionally active double-layered

particles; in the model shown as a spike-less rough blue particle) through a raft-mediated process. In this minuet performance, the NA-resistant mutant nar3 enters the dance

by interacting with integrin a2b1. The entry point for naturally occurring NA-resistant strains could be either a NA-resistant SA-containing molecule (i.e. GM1) or a non-acidic

sugar residue (i.e. galactose). However, integrin a2b1 has also been suggested to be the binding receptor for both NA-sensitive and -resistant rotavirus strains [12,22]. There

remains a question mark on how particles penetrate the plasma membrane and how they loose the outer layer to become transcription-active double-layered particles.

β1hsc70

α2 β2αxβ3αv


Cell membraneSialoreceptor

VP8 VP5

Lipid raft in thecell membrane

?

VP5 VP7

VP5

VP8VP7

VP7




nine amino acids that showed 67% identity between RRVVP7 and the G1G2 protein of hantavirus L99. Thissequence in VP7 is highly conserved among differentrotavirus strains, and a synthetic peptide of this region(peptide CNP) has been shown in vitro to bind to avb3using a different site to the RGD-binding site, and toefficiently block the infectivity of rotavirus RRV strain butnot its binding to the cell (S. Zarate, et al., unpublished).Altogether, these results indicate that rotaviruses interactwith avb3 through the CNP region of VP7, which is locatedbetween amino acids at positions 161 and 169.

Integrin axb2–VP7

Further analysis of the reassortant viruses describedabove indicated that VP7 also interacts with integrinaxb2. This is consistent with the observation that VP7contains the axb2 integrin ligand sequence GPR posi-tioned at amino acid residues numbered 253 to 255 [12].Characterization of the axb2–VP7 interaction showedthat the synthetic peptide GPRP blocked the infectivity ofthe rotavirus strains RRV and Wa but not their cell-binding, confirming that, as previously suggested [23], theinteraction of the virus with this integrin also occurs at apost-attachment step. In agreement with these findings, ithas been described that antibodies to VP7 neutralizerotavirus infectivity by blocking a post-binding event [42].

Role of lipid rafts in virus cell entry

The infectivity of rotaviruses is partially blocked bymetabolic inhibitors of N-glycosylation and glycolipidsynthesis, and is also severely impaired by the depletionof cholesterol from the cellular membrane [43]. On thebasis of these findings it was suggested that sphingolipid-and cholesterol-enriched membrane lipid microdomains,usually referred to as lipid rafts (Box 2) [44], might beinvolved in the entry of rotaviruses into the cell [27,43,45].The participation of lipid rafts in rotavirus infection issupported by the observation that ganglioside GM1,integrin subunits a2 and b3, and hsc70 are all associatedwith these lipid microdomains and that infectious particlesalso associate with rafts during early interactions of thevirus with the cell [46]. These results suggest that rafts, ashas been found for other viruses [47], might play animportant role in the cell entry of rotavirus, possiblyserving as platforms to facilitate the efficient interaction ofcell receptors with the virus particle.

Mechanism of rotavirus cell entry

Early electron microscopy studies of rotavirus-infectedcells suggested endocytosis as the virus internalizationpathway. However, it was later shown that rotavirusinfectivity is not inhibited either by preventing theacidification of endosomes or by drugs that block theintracellular traffic of endocytic vesicles [9]. Direct cellmembrane penetration has also been postulated as themechanism of virus entry on the basis of electronmicroscopy data and on the observation that rotavirusinfection induces a rapid permeabilization of the cellmembrane [9]. A hydrophobic domain of VP5 thatpermeabilizes model and bacterial membranes has beensuggested to be involved in this process [48,49].

Recently, a calcium-dependent endocytosis model wasproposed for rotavirus entry on the basis of the observationthat solubilized VP4 and VP7 are able to permeabilizemembranes [50] and that bafilomycin A, a drug thatinhibits the endosomal proton-ATPase pump, blocksrotavirus infection [51]. In this model, the virus isinternalized within clathrin-coated endocytic vesicles;within these vesicles a decrease in the calcium concen-tration, promoted by an endosomal proton-ATPase pump,solubilizes the surface proteins from the virus particle. Thesolubilized outer layer proteins then permeabilize thevesicle’s membrane to release the transcriptionally activedouble-layered particle into the cytosol [51]. However, ithas been recently reported that drugs and dominant-negative mutants that are known to impair clathrin- andcaveolae-mediated endocytosis did not affect rotavirus cellinfection, whereas cells expressing a dominant-negativemutant of dynamin, a GTPase known to function in severalmembrane scission events, were not infected by the virus[52]. These results, together with the observation thatdepletion of cholesterol inhibits rotavirus infection,suggest that rotaviruses might use a recently definedcell internalization pathway to enter cells. This pathway isdescribed as raft-dependent endocytosis and is defined byits clathrin and caveolin independence, its dependence ondynamin and its sensitivity to cholesterol depletion [53].

In addition to its property of severing membranes,dynamin has been recently implicated in numerousprocesses that involve membrane dynamics, such as theformation of podosomes, vesicle comet motility, and actinrearrangements and cytokinesis [54]. Therefore, thedependence of rotavirus entry on functional dynamin

Box 2. Lipid rafts

Cellular lipid rafts are membrane lipid microdomains enriched in

glycosphingolipids (gangliosides among others), cholesterol and a

specific set of associated proteins. The preponderance of saturated

hydrocarbon chains in cell sphingolipids allows for cholesterol to be

tightly intercalated, similar to the organization of the liquid-ordered

state in model membranes. The lipids in these domains differ from

other membrane lipids in their lateral diffusion on the membrane and

can be physically separated by density centrifugation given their

insolubility in some detergents [67].

Rafts have been implicated in a variety of cellular functions:

† Apical cell-sorting of proteins.

† Signal transduction.

† Caveolae-mediated endocytosis.

Rafts are also used by viruses as:

† Assembly and budding sites in the cell membrane (e.g. influenza

virus, HIV, measles virus, rotavirus).

† Intracellular trafficking of viral proteins (e.g. echovirus types 1 and

11).

† Platforms for cell entry (e.g. HIV, SV40, echovirus type 1 and

rotavirus).

Rafts can be disrupted experimentally to evaluate their role in

specific biological phenomena or functions.

This is accomplished by removing or sequestering the cholesterol

from the plasma membrane, without compromising the viability of

the cell. The most commonly used drugs are:

† Nystatin and filipin (sequester cholesterol).

† Methyl-b-cyclodextrin (depletes cholesterol).

† Lovastatin (inhibits biosynthesis of cholesterol).




might be the result not only of its participation in theendocytosis pathway per se, but it could also be involved ata later entry step during the movement of the virus fromthe plasma membrane to the cytosol. It is also important tomention that to date it has not been ruled out thatrotaviruses could possibly enter the cell at the plasmamembrane level, using a non-defined, direct entry mech-anism, in which the depletion of cholesterol could eitheralter the lateral mobility of plasma membrane proteins[55] or disrupt the organization of lipid rafts that might beholding together the rotavirus receptors and impairingvirus entry.

Concluding remarks

The recent advances in understanding the early inter-actions of rotavirus with its host cell have been fueled bythe identification of rotavirus cell receptors. Progress inunderstanding plasma membrane organization, the defi-nition of different types of endocytosis and the applicationof cryo-electron microscopy to study virus structure, havealso been crucial for this purpose. The data presented hereindicate the existence of several rotavirus receptors, someof which interact sequentially with different domains ofthe virus surface proteins and might be tightly organized,possibly forming a complex, in glycosphingolipid-enrichedrafts (Figure 3). The requirement of several cell molecules,which need to be present and organized in a precisefashion, might explain the selective cell and tissue tropismof these viruses. Future research (Box 3) should define therole that each individual receptor plays in virus entry, theconformational changes experienced by the viral proteins

upon interaction with these receptors and the mechanismof virus internalization. Of utmost importance will be thedetermination of the receptors and the entry pathway usedby rotaviruses to infect mature enterocytes in a naturalinfection. A combination of standard biochemical andmolecular tools, together with the use of animal modelsand new technologies, such as RNA interference, as well asthe continued application of high-resolution structuralcryo-electron microscopy and X-ray crystallography ofviral proteins and receptors, will be required to gaininsight into the elaborate mechanism used by rotavirusesto enter cells.

AcknowledgementsWe would like to thank U. Desselberger for critical reading of themanuscript and for valuable suggestions and comments, and also P. Isaand E. Mendez for helpful discussions on the manuscript. We appreciatethe support by B.V.V. Prasad for providing the cryo-electron microscopyimages of the virus. We apologize to colleagues whose work has not havebeen cited in full owing to length constraints. Work on rotavirus cell entryin our laboratories is supported by grants 55003662 and 55000613 fromthe Howard Hughes Medical Institute and G37621N from the NationalCouncil for Science and Technology-Mexico.

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Box 3. Questions for future research

† Is the binding to sialic acid (SA) an essential step for rotavirus

infection in vivo?

† Which is the primary attachment receptor for neuraminidase (NA)-

resistant rotavirus strains?

† Do integrins need to be activated to mediate rotavirus infection?

† Does the interaction of the virus with integrins induce a signaling

cascade necessary for virus infection?

† Is the chaperone activity of the heat shock protein hsc70 needed for

rotavirus entry?

†What is the individual contribution of each of the cellular molecules

involved in rotavirus infection to the permissive status of a cell? Are

all the receptors needed for an efficient virus infection or do they

represent alternative pathways for cell entry?

† Is there a unique pathway of infection for all rotavirus strains, with

distinct entry points for different strains?

† What are the conformational changes that the viral proteins

undergo during virus entry?

† What is the mechanism by which the trypsin cleavage of VP4

mediates rotavirus entry?

† What are the role of rafts in rotavirus entry?

† Where does the virus particle uncoat – within an endocytic vesicle

or during the transit of the virion through the cell membrane?

† What is the role of dynamin in rotavirus internalization?

† What are the receptors and the entry pathway used by rotaviruses

in a natural infection of mature enterocytes in the gut?

† Is receptor blockage a rational and feasible way to develop an

antiviral drug?

† Will the conformational changes in the viral proteins that are

predicted to be induced by their interaction with cell receptors

expose new domains that could be targeted with neutralizing

antibodies or antiviral drugs?




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multistep entry of rotavirus into cells: a versaillesque dance

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