Structural Analysis of Human Rhinovirus Complexed with ICAM-1 Reveals the Dynamics of Receptor-Mediated Virus Uncoating

Intercellular adhesion molecule 1 (ICAM-1) functions as thecellular receptor for the major group of human rhinoviruses,being not only the target of viral attachment but also the mediatorof viral uncoating. The configurations of HRV3-ICAM-1 complexesprepared both at 4°C and physiological temperature (37°C)were analyzed by cryoelectron microscopy and image reconstruction.The particle diameters of two complexes (with and without RNA)representing uncoating intermediates generated at 37°C wereeach 4% larger than that of those prepared at 4°C. The largervirus particle arose by an expansive movement of the capsidpentamers along the fivefold axis, which loosens interprotomercontacts, particularly at the canyon region where the ICAM-1receptor bound. Particle expansion required receptor bindingand preceded the egress of the viral RNA. These observationssuggest that receptor-mediated uncoating could be a consequenceof restrained capsid motion, where the bound receptors maintainthe viral capsid in an expanded open state for subsequent genomerelease.

INTRODUCTION

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Introduction

Human rhinoviruses (HRVs), a genus of the picornavirus family,are the major cause of the common cold in humans (25). Theyconstitute a group of approximately 100 serotypes, all containinga single-stranded RNA genome encapsulated into an icosahedralcapsid. Crystal structures of several rhinovirus serotypes demonstratedthat the capsid is made of 60 protomers, each having a copyof four structural proteins (VP1 to VP4) (10, 24, 29). The threemajor proteins (VP1, VP2, and VP3) build the capsid shell. Theyhave a central "jelly roll" structure, with their N-terminalregions being located close to the internal VP4 and RNA genome.Biochemical experiments showed that VP4 and internal N-terminalresidues of VP1 were accessible to immobilized trypsin in HRVserotype 14 (HRV14), revealing that they became exposed spontaneouslyoutside of the capsid (17). These findings demonstrated thatthe capsid shell of HRV is a dynamic, breathing entity.

The most highly conserved structural feature among HRVs is asurface depression around the fivefold axes, the so-called canyon(25). The canyon is composed of the north wall (built by VP1)around the fivefold axis and the south wall (built mainly byVP2 and VP3). While the most accessible residues along the canyonwalls are hypervariable, the less exposed residues in the canyonfloor are conserved and used for receptor binding, as the strategyallowing the virus to escape antibody neutralization (23). Thereis a hydrophobic pocket in VP1 directly underneath the canyonfloor. The pocket appears empty in HRV3 and HRV14 (29) but isfilled with electron density resembling a fatty acid (pocketfactor) in the structures of HRV2 and HRV16 (10, 27). The pocketfactor stabilizes the virion during its cell-to-cell transit(19). Hydrophobic antiviral compounds (WIN compounds) were foundto bind to the VP1 pocket and to inhibit capsid breathing (17).

The major group of HRVs use the canyon region for binding toits cellular receptor, intercellular adhesion molecule 1 (ICAM-1)(16). ICAM-1 is a membrane protein with five immunoglobulin-likedomains in the extracellular region. The critical virus bindingepitopes are present at the tip of the N-terminal first domain(D1) of ICAM-1 (16), which penetrates into the rhinovirus depressivecanyon (2, 16). The low accessibility of the receptor bindingsite in the major group of HRVs correlates with a low virus-receptorassociation rate (28). Binding of ICAM-1 to HRV3 and HRV14 (receptor-sensitiveserotypes) triggers the release of the RNA genome from the capsid(uncoating), but this does not occur with other members of themajor group of HRVs (13, 28). Receptor-mediated uncoating hasbeen reported for the major group of HRV and poliovirus, showingthat these viruses use their receptors for both virus attachmentand uncoating (13, 15, 21). Indeed, entry of poliovirus andreceptor-sensitive rhinoviruses (HRV14) can occur without endosomalacidification (20, 26).

The structural changes mediated by the receptor in the virusparticles lead to the release of the RNA genome from the capsidfor penetration into the cell cytoplasm (13, 15, 21). It hasalso been proposed that the RNA will move through a membranepore generated by the externalized N-terminal region of VP1and VP4 during the uncoating process (3). Externalization ofthose polypeptides has been supported by two structural analysesof high-temperature (>50°C)-treated poliovirus and aminor-group rhinovirus, which have provided new insights intopicornavirus uncoating (3, 12). However, no structural analysisof receptor-mediated uncoating has been reported so far, incontrast to the well-documented biochemistry of this process(5, 13, 15). The precise mechanism by which a cellular receptortriggers the exit of the viral genome is currently unknown.To gain new insight into this process, we applied cryoelectronmicroscopy (cryo-EM) to a well-defined model, HRV3 and its ICAM-1receptor (5, 13). Our results indicate that the receptor catalyzesuncoating by locking the viral capsid in an expanded open statefor subsequent RNA release.

MATERIALS AND METHODS

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Materials and Methods

Preparation of receptor-virus complexes and uncoating intermediates. The receptor-HRV complexes were prepared by incubating purifiedvirions with a two-domain fragment of ICAM-1 overnight at 4°C(28). Unbound receptor was removed via filtration through a1-ml Superose-12 column in 20 mM Tris-HCl buffer (pH 8.0) with10 mM MgCl₂ and 50 mM NaCl. Fractions containing the complexeswere determined by measuring the absorbance at 260 nm and wereused to prepare vitrified specimens for cryo-EM. After overnightincubation, the H3R^C sample was directly subjected to the gelfiltration step at 4°C; the other complexes were furtherincubated at 37°C for 15 and 60 min for HRV3-ICAM-1 andHRV16-ICAM-1 complexes, respectively. These samples were thenchilled on ice for 5 min before removal of unbound receptorsat 4°C.

Structure determination and analysis. A Philips CM120 microscope equipped with a Gatan cryotransfersystem was used to photograph the HRV-receptor complex. Themicrograph then was digitized at 3.1 Å/pixel on a SCAIscanner. Images of individual particles were boxed out for furtheranalysis based on icosahedral symmetry (7). The data from thelarge defocus film was processed first to get a stable modelmap for the close-defocus data. The H3R^C complex was reconstructedto a resolution of 23 Å by combining 125 individual close-defocusimages. The H3R^C reconstruction served as the initial modelto assign the center and the orientation of the particles at37°C (H3R^H and H3Re). The single-image reconstructions wereused to aid the particle selection and to help in fine-tuningthe intermediate polar Fourier transform models (1). The sizeof individually boxed H3R^H images was determined by correlatingthe observed data with the corresponding projection of the H3R^Creference map by grid interpolation. The Gaussian distributionof the particle size of H3R^H versus H3R^C was shifted, with a4% increase in the mean value. The final reconstruction of H3R^Hand H3Re complexes was resolved to 23 and 25 Å by using91 and 64 particles, respectively. The resolution was determinedby R-factor assessment. The completeness of the data samplingin the reconstruction was verified by obtaining eigenvalue spectra,showing that of eigenvalues were >1 or 10 (8).

In a particular experiment, the internal size standard was generatedby the addition of a large portion of native HRV3 particlesto the 37°C-treated HRV3-receptor mixture after it was chilledon ice. The empty HRV3-receptor complex and full particles,mainly including unheated HRV3, were boxed out from a singlemicrograph and were used to calculate three-dimensional maps.Comparison of these three-dimensional reconstructions gave 4%size difference. The consistent dimensions of the bound ICAM-1molecule ( $~$ 75 Å long and $~$ 20 Å in diameter) in thethree complexes demonstrated no detectable systematic errordue to the microscope. Finally, the consistent size of the HRV16-receptorcomplexes at different temperatures was an additional controlfor the collection of low-dose imaging of microscope data.

Model fitting. Fitting of the ICAM-1 receptor (PDB access number 1IC1) andthe HRV3 crystal structures (PDB access number 1RHI) into thecryo-EM density was performed manually using the program O (14).The docking of coordinates was done based on both visual andquantitative criteria. The HRV3 coordinates and H3R^C map hadthe same origin. Domain 2 of the ICAM-1 molecule first was fittedinto the corresponding EM density, with a previously describedorientation as reference (16). The position of receptor domain1 was adjusted manually. To model the capsid movement in theH3R^H structure, the alpha-carbon coordinates of ICAM-1 werefitted into the receptor density of the expanded particle andthen the coordinates of the viral proteins were fitted individuallyinto the H3R^H map. VP2 and VP3 were moved out radially and tiltedto best follow the density distribution. Based on the modeledICAM-1 position with its unique density profile, contact betweenVP2 and ICAM-1 was maintained and was used to localize the positionof VP2. Subsequently, VP1 was docked into the EM density beforethe position of the whole protomer was adjusted by minimizingthe crash with neighboring protomers. The resulting model wasused to calculate a set of structure factors from which an electrondensity map was generated at a resolution corresponding to theEM density. The real-space correlation between the calculatedand experimental densities was computed, and the high valueof the correlation coefficient in the H3R^H fitting (0.79) indicatedoptimal positioning of the viral proteins. This value was similarto that of H3R^C docking (0.80). The best correlation coefficientof H3Re fitting was 0.73 (data not shown).

RESULTS

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Results

Reconstruction of HRV3 entry intermediates. Receptor binding to HRV3 at physiological temperature generatesvirus-receptor intermediates, leading to the simultaneous exitof the internal RNA and VP4 and the subsequent appearance ofempty viral capsids (5) (Fig. 1A). Cryo-EM micrographs showedthat while HRV3 and ICAM-1 formed stable complexes at 4°C(H3R^C) (Fig. 1B), a mixture of full (H3R^H) and empty (H3Re)particles appeared after a 15-min incubation of the H3R^C complexesat 37°C (Fig. 1C). About 40% of the particles were emptyfor HRV3, as judged by their lower density in the center ofthe capsid. In contrast, no empty particle was observed forthe stable HRV16 even after 60 min of extensive incubation withICAM-1 at 37°C (data not shown), consistent with the inabilityof the receptor to mediate uncoating of this serotype at neutralpH (13).

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FIG. 1. Cryo-EM of HRV3-receptor complexes and uncoating intermediates. (A) Schematic view of the pathway of HRV3 uncoating mediated by soluble ICAM-1 binding (5) and the procedure used for preparation of virus-receptor complexes for cryo-EM. Arrows and hexagons represent the ICAM-1 receptor and the HRV particles, respectively. H3R^C was prepared by incubation of HRV3 with ICAM-1 at 4°C, while H3R^H and H3Re were prepared at 37°C as described in Materials and Methods. H3R^C and H3R^H preserve all viral proteins and RNA, while H3Re lacks both VP4 and RNA (5). (B) Micrograph of H3R^C. (C) Micrograph of H3R^H and H3Re complexes. About 40% of the particles appear empty (H3Re), lacking the internal RNA genome represented by a spiral line in panel A. Bar, 1,000 Å.

Three-dimensional reconstruction of the H3R^C complexes (Fig.2) revealed binding of the ICAM-1 receptor perpendicularly tothe viral capsid into the canyon region around the 12 icosahedralfivefold axes (Fig. 2). Receptor binding occupancies were similarin the H3R^C, H3R^H, and HRV16-receptor (not shown) complexesand higher than in the H3Re particles. The capsid distal densityof ICAM-1 corresponding to domain 2 vanished in the 3.0- ${sigma}$ -contouredmap of H3Re. However, disappearance of the EM density of thatICAM-1 region in the H3R^C and H3R^H maps required contour levelsof 3.5 and 3.8 ${sigma}$ , respectively. These data showed that the receptormolecule remained bound to the capsid during RNA release andsuggest that the bound receptor slowly released from the capsidafter the egress of RNA (5). The lower occupancy of ICAM-1 inH3Re could be also due to a less ordered bound receptor molecule.

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FIG. 2. Surface rendering of native HRV3 and virus-receptor complexes. The maps are viewed along the twofold axis and contoured at 1 ${sigma}$ above the average. The labels of the complexes correspond to the data acquisition scheme presented in Fig. 1. In the right column, maps truncated 10 Å below the surfaces at the fivefold axis are shown. The locations of icosahedral two-, three-, and fivefold axes are indicated by the numbers superimposed on the HRV3 reconstruction, which was computed from the crystallographic coordinates (29). Bar, 100 Å.

The star-shaped fivefold plateau in H3R^H is very similar insize and shape to that of H3R^C and differs from the broaderplateau presented by H3Re (Fig. 2, right). Unlike in H3R^C, wherea clear boundary between the receptor and the fivefold plateauis quite apparent (Fig. 2, right), the receptor appears connectedto the side of the plateau by a protruding density in H3R^H (seealso Fig. 5). The bound receptor underwent a pronounced clockwiserotation of approximately 30° after RNA release in H3Re,so that the long axis of the receptor footprint was parallelto the edge of the plateau (Fig. 2, bottom right).

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FIG. 5. Virus-receptor binding surfaces. Side (top) and front (bottom) views of the fitted ICAM-1 and HRV3 crystal structures into the corresponding EM densities (grid) of the H3R^C (A and D) and H3R^H (B and E) complexes, superimposed in panels C and F. Density maps are contoured at 1.5 (top) and 2.0 (bottom) ${sigma}$ . A ribbon diagram of the two-domain crystal structure of ICAM-1 is shown, with the two interacting HRV3 protomers presented as worm drawings. The two connecting viral protomers are colored in yellow (left protomers in panels A, B, D, and E) and magenta (right protomers). In panels C and F, viral proteins VP1, VP2, and VP3 of H3R^H are colored in blue, green, and red (the left protomer) or magenta (the right protomer), respectively; the superimposed H3R^C protomer is colored in yellow. VP4 is cyan, and the N-terminal 60 residues of VP1 are gray. The red arrow points to the protruding density featuring the H3R^H particle, predicted to be filled by the externalized VP4 and the VP1 N terminus. The side chain of the VP3 Tyr181 residue is indicated by purple spheres. The first (D1) and second (D2) domains of ICAM-1 are labeled, as are virus-binding BC, DE, and FG loops (6). The illustrations were prepared with the program PyMOL (http://www.pymol.org).

Capsid expansion precedes RNA release. Reconstruction of HRV3-receptor complexes revealed differencesin size between the viral particles exposed at 37 and 4°C(Fig. 2, right). The virus particle in H3R^H expanded approximately12 Å in diameter compared to that in H3R^C (Fig. 3, top),even though this complex was reported to preserve all the viralproteins and the RNA genome (5). As a result, the C-terminalend of ICAM-1 moved radially from 216 to 222 Å in H3R^Hwhile the length of the receptor remained essentially the same.The distance between the end of the receptor density and thecapsid fivefold axis for H3R^C and H3R^H was 49 and 48 Å,respectively. Similar expansion was observed for H3Re (datanot shown). Capsid expansion was unique for the HRV3-receptorcomplexes since it was not observed for HRV16-receptor complexesor free HRV3 virions exposed at physiological temperature (Fig.3). Because uncoating was also specifically observed in theRNA-containing HRV3-receptor complex, we concluded that expansionprecedes the egress of the viral genome.

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FIG. 3. Size of the virus particles in the HRV-receptor complexes. Density profiles of the HRV reconstructions, presented as spherically averaged density versus particle radius, are shown. The arrows indicate the outer boundary of the viral capsid. (Top) Density distribution for the H3R^C and H3R^H reconstructions presented in Fig. 2. (Middle) Density distribution for HRV16-ICAM-1 complexes prepared at 4°C (H16R^C) or 37°C (H16R^H) as described in Materials and Methods. (Bottom) Density distribution for HRV3 native virions treated at 4°C (HRV3^C) or 37°C (HRV3^H) in the absence of receptor.

Capsid rearrangements in the HRV-receptor complexes. Capsid expansion implies conformational rearrangements in theviral particles. An equatorial section of the particles showeda less compact RNA in the interior of H3R^H than H3R^C and anempty shell in the H3Re (Fig. 4). Lower density was observedin the two uncoating intermediates across the junction of twofoldrelated protomers (red contour line in Fig. 4B and C). A significantdecrease in the capsid density at the canyon region was observedin the H3R^H and H3Re complexes compared to H3R^C and appearedas a "density cleft" near the receptor binding site in the high-contourmaps (asterisk in Fig. 4). The canyon region is built up bytwo neighboring protomers (see below), so the decrease of EMdensity in that region could be related to separation of theprotomers in the expanded virus-receptor complexes.

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FIG. 4. Capsid rearrangements in the HRV-receptor complexes. (A to C) Equatorial sections of H3R^C (A), H3R^H (B), and H3Re (C) reconstructions perpendicular to the icosahedral twofold axis. The icosahedral axes (two-, three-, and fivefold) are drawn on the section of H3R^C. The contour plots circle the density distribution at 1 ${sigma}$ (blue) and 2 ${sigma}$ (red). The arrow in panel C indicates the twist movement predicted for VP1. (D) Top view of the H3R^C and H3Re electron density maps of a viral pentamer. The H3Re density map (cyan grid lines) contoured at 2 ${sigma}$ is superimposed onto the H3R^C isosurface map rendered at 3 ${sigma}$ . The red line indicates the location of the equatorial sections presented in panels A to C. An asterisk marks the canyon position. The illustrations were prepared with the NAG Explorer program.

The exit of the viral genomic RNA led to major rearrangementsat the fivefold plateau and VP1 (Fig. 4C). The canyon northwall appears more parallel to the fivefold axis in H3Re thanH3R^C-H, suggesting a pronounced twist movement of VP1 (red arrowin Fig. 4C). As a consequence, the plateau became broader inthe empty complex (also shown in Fig. 2).

Receptor-virus binding contacts. The crystal structure of the two N-terminal domains of ICAM-1fits well into the corresponding density of the H3R^C and theH3R^H maps (Fig. 5A and B). The receptor appears more bent inH3R^H than in H3R^C (Fig. 5C). The side of the first domain (ß-strandsC and F) contacts the south wall of the canyon (the promoteris shown in yellow), and the tip (BC and FG loops of domain1) penetrates into the canyon and sits on the junction of twoclosely associated promoters (Fig. 5D and E).

In H3R^H, the neighboring promoters remain in contact at thefivefold axis region but are separated in the canyon (Fig. 5E);this is due mainly to the expansion of the capsid and to theslight movement of VP2 and VP3 toward the threefold axis (Fig.5F). The docking of the viral proteins in H3R^H is consistentwith the preservation of the density organization at the fivefoldaxis as well as with the appearance of a density cleft at thecanyon (Fig. 2 and 4) concomitant with capsid expansion. Wehave observed that residues such as Tyr181 of VP3 become exposedat the interprotomer junction of the fitted viral proteins inthe expanded capsid. This residue could then interact with thecritical Pro70 residue located in the FG loop of the receptor(Fig. 5B and E).

The internal N-terminal regions of VP1 and VP4 are located underneaththe hole at the canyon region generated by capsid expansion.Those polypeptides then could externalize through this regionand account for the protruding density connecting the receptorto the north wall of the canyon in H3R^H (arrowhead in Fig. 5Band C). The externalized proteins may interact with the relativelyhydrophobic DE loop of ICAM-1, which is involved in crystallographicdimeric association (6).

DISCUSSION

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Discussion

Complexes of the ICAM-1 molecule with HRV3 and HRV16 were analyzedto elucidate how receptor binding mediates subsequent capsiduncoating. Our results provide the first structural view ofvirus uncoating mediated by receptor binding under conditionssimilar to those found at the cell surface. Capsid expansionin receptor-bound HRV3 was observed in conjunction with uncoating.However, no such changes were observed either with HRV3 virusestreated at 37°C in the absence of receptor or with the stableHRV16-receptor complex. These observations suggest that uncoatingrequires receptor-mediated capsid expansion, which breaks interprotomerconnections for externalization of capsid proteins and RNA (schemein Fig. 6). The expansion observed here was similar to thatreported for the 135S entry intermediates of poliovirus generatedby heating (3), suggesting that viral capsid expansion may precedeRNA release during virus entry.

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FIG. 6. Dynamics of receptor-mediated rhinovirus uncoating. Schematic views of two neighboring protomers with a bound ICAM-1 receptor molecule, representing conformations of H3R^C, H3R^H, and H3Re complexes. Ovals, triangles, and pentagons indicate the positions of the twofold, threefold, and fivefold axes, respectively. VP1, VP2, and VP3 are represented as blocks colored blue, green, and red, respectively. Stable virus-receptor complexes (H3R^C) are formed at low temperatures by receptor binding to a close virus conformation, represented by the crystal structure (28). The capsid dynamics (or breathing) opens the interprotomer junction, so that the internal N-terminal regions of VP1 and VP4 (labeled as N-VP1-VP4) became exposed simultaneously at physiological temperatures (17, 18, 22). This expanded conformation is maintained by receptor binding between two adjacent protomers and to the exposed region of VP1, as shown by the cryo-EM H3R^H structure. Egress of the viral RNA genome and loss of VP4 result in major rearrangements in VP1 and the fivefold axis and subsequent release of the receptor from the H3Re complex.

The pocket factor molecule present in HRV16 appears to inhibitboth capsid dynamics and uncoating (17). These data, along withthe temperature dependence of rhinovirus uncoating (5, 13),suggest that capsid breathing might initially drive particleexpansion (Fig. 6). Therefore, rather than being the trigger,the bound receptor molecules would just hold the capsid in anexpanded conformation by binding within two neighboring capsidprotomers in their expanded open state (Fig. 5 and 6). Our modelimplies receptor binding to several conformations of the canyonregion, as suggested by the nonlinear kinetic binding rates(28).

A protruding and well-defined density appeared, bridging thereceptor and the north canyon wall in the expanded H3R^H capsid,but it was absent in H3R^C and H3Re (Fig. 2 and 5). This densitymight represent the externalized N-terminal regions of VP1 andVP4, which have been reported to exit simultaneously and reversiblyfrom the capsid (17, 18). These polypeptides localize beneaththe interprotomer interface in the native capsid (Fig. 5D).Thus, these two peptides may exit through the open canyon regionand account for the protruding density featuring the H3R^H intermediate.Externalization of these polypeptides through a similar locationhas been proposed for related picornaviruses (3, 12). Localizationof the externalized polypeptides near the hydrophobic ICAM-1dimerization region (6) suggests that they may interact withthe receptor. This interaction could lock the VP1 N terminusand VP4 in their externalized conformations so that they couldbind to the membrane (9).

The release of the RNA from the capsid of picornavirus is thoughtto occur through one of its 12-pentameric vertices. However,there has been no evidence showing the egress of the VP3 N-terminalcylinder connection, VP1 N terminus, VP4, or viral RNA throughthis opening. Analyses of uncoating intermediates in HRV3 presentedhere and in poliovirus (135S) (3) showed little evidence forthe formation of a capacious tunnel through the fivefold axisregion. Changes at the fivefold axis region arise after RNAegress and might lead to capsid disassembly (Fig. 2 and 4) (12).

While the fivefold axis regions of H3R^C and H3R^H particles arestructurally similar, significant differences were observedalong the interprotomer interfaces in the canyon region andat the twofold axis junctions (Fig. 2 and 4). These observationsstrongly suggest an expansive movement of the pentamers alongthe fivefold axis. Polarized expansion of a single receptor-attached,membrane-proximal pentamer could occur during virus entry. RNAegress through the expanded pentamer could then take place closeto the membrane while the virus particle remains attached tothe receptor, thereby facilitating penetration into the cellcytoplasm.

The results presented here reveal for the first time that capsidexpansion can be mediated by receptor binding at physiologicaltemperature. The magnitude of capsid expansion for the H3R^H(4%) uncoating intermediate is similar to that observed forrelated picornavirus particles (3, 12), so that it must be significantfor virus entry. We propose that the ICAM-1 receptor passivelycatalyzes uncoating by locking the capsid in an expanded andopen intermediate state for subsequent RNA release (Fig. 6).In rhinovirus and related picornaviruses, this process can beattributed to the dynamic feature of the viral capsids (17,18, 22), as well as the fragmented nature of the receptor bindingsite (4, 11, 16, 28). Expansion of nonenveloped virus capsidscatalyzed by receptor binding or other cellular factors couldprecede uncoating during virus entry into cells, as shown herefor HRV and its ICAM-1 receptor.

ACKNOWLEDGMENTS

We are grateful to Birgitta Lindqvist for assistance with tissueculture. We thank Lars Liljas, Henrik Garoff, and Maarit Suomalainenfor helpful suggestions about manuscript preparation.

This work has been sponsored by grants from the Medical ResearchCouncil (MFR-12175 and MFR-12637) and the Natural Science ResearchCouncil (NFR-11691 and NFR-11994) to R.H.C. and J.M.C. L.X.is the recipient of a fellowship from the Swedish StructuralBiology Network (to R.H.C.).

FOOTNOTES

* Corresponding author. Mailing address for Li Xing: Department of Biosciences, Karolinska Institute, 141 57 Huddinge, Sweden. Phone: 46 8 6089123. Fax: 46 8 7745538. E-mail: li.xing{at}biosci.ki.se.

Present address for José M. Casasnovas: Centro Nacionalde Biotecnología, CSIC, Campus Universidad Autónoma,28049 Madrid, Spain. Phone: 34 91 5854917. Fax: 34 91 5854506.E-mail: jcasanovas@cnb.uam.es.

REFERENCES

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