Rotavirus spike structure and polypeptide composition.

0022-538X/91/084334-07$02.00/0

CopyrightC) 1991, AmericanSociety forMicrobiology

Rotavirus

Spike Structure and Polypeptide Composition

INDUMATHY D. ANTHONY, STANLEYBULLIVANT, SHOBHNADAYAL, A.RICHARD BELLAMY,* ANDJOHN A. BERRIMANt

Departmentof Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand Received 29 November1990/Accepted 10 May 1991

Negativelystainedpreparationsof rotavirusimagedwithalowdose of electronsprovidesufficientcontrast toreveal surfaceprojectionsorspikes.The number ofspikesfoundprojectingfrom differentparticles indicates thatnot all 60 peripentonalsites are occupied. Treatment atpH 11.2 with 250 mM ammonium hydroxide specificallyremovesthe spikes, yieldingsmooth double-shelled particles of the samediameterasthat ofthe native virus. Protein analysis confirmsthat the released spikesarecomposed ofpolypeptide VP4(orits two cleavageproducts VP5* and VP8*)and that thesmooth particle retains theothermajoroutershell protein VP7.Spikeless particles canbedecoratedby amonoclonalantibody specific for themajorimmunodominant

neutralizingdomainof VP7, implyingthat removalofthespikesdoesnotdenature the VP7that is retainedon

thesurfaceof the smoothparticle.

The outershell of thedouble-layered rotavirus particle is constructed fromtwo viral proteins. The most abundant of these is VP7, a 38-kDa glycoprotein which is the

type-specific antigen (9, 12). VP7 is the translational product of genomic segment 9 (3, 17). The second protein, VP4 (88 kDa), is encoded by genomic segment 4 and is the viral hemagglutinin, a protein which also specifies cell tropism (9). Both VP7 and VP4 are assembled on the surface of the

single-shelled rotavirus particle at some stage during the maturation of the virus in thelumen ofthe rough

endoplas-mic reticulum (20, 21). However, when trypsin is incorpo-ratedintotheculture medium, VP4is cleaved proteolytically to yieldVP5* (60 kDa)and VP8* (28kDa), anevent which greatly enhances infectivity in vitro (4, 6, 7). The cleavage sitesprobably resideatArg-241and Arg-247, residues which areconserved in most strains of rotavirusfor whichcDNAs have been sequenced (2).

The capsomeres of the inner shell have been shown by platinum shadowing to be arranged in a T = 13L configura-tion (19, 24). Prasadetal. (23) appliedcryoelectron micros-copy and image-averaging techniques to construct a model for therotavirus particle with aresolution of 4.0nm. Their work revealed that the outershell is also arranged in aT =

13L symmetry and that there are 60 surface projections

(spikes) approximately4.5 nmlong and 3.5nmwide. They

estimatedfrom their model that eachspike was one molecule of VP4. In a subsequent analysis (22), the spike was

de-scribed as athin structure extending to a height of 4.5 nm, withafurther well-defined globular domain of approximately 5.5nmindiameter, acrosswhich lies abilobedstructure4.0 nmwide and 7.0nmacross. The estimated mass of the spike and the fact that two Fab fragments appeared to bind to each spike led these researchers to infer that the spike was a dimer of VP4.

Yeager et al. (26) have also reconstructed images of rotavirus from cryoelectron micrographs and similarly de-scribe the spike as having a complex bilobed morphology. The spikes were found to be shorter by the reconstruction method than could be measured on the micrograph, and they

* Correspondingauthor.

tPresent address: Laboratory of Molecular Biology, Medical ResearchCouncil, CambridgeCB22QH, England.

suggested that morphological variability would be lostas a result of the averaging implicit in the reconstruction proce-dure. Indiscussing the oligomeric nature of the spike, they drew attention to the large disparity of stoichiometry be-tween VP4 and VP7 from densitometer measurements of stainedpolyacrylamide gels (15) and suggested thatpurified viruses may well not all carry the full complement of 60 spikes. In a study of rotavirus by the freeze-etch method (la), it was found that while spikes on the surface of the intact virus could beclearlydemonstrated, occupancy of the peripentonal sites was low.

Here we present new evidence on the nature of thespikes by using negatively stained preparations examined with a low dose (26) of electrons (low-dose images). This method has enabled us totest many different extraction conditions for their effect on the virus. We have found that treatment of theintact virion with the weak base ammoniumhydroxideat pH 11.2 removes the spikes, releases VP4 (or its cleavage products VP5* and VP8*) into the supernatant, and yields smoothparticles that are otherwise intact.

MATERIALSANDMETHODS

Cellsand virus. The SAl1 strain ofrotavirus was

propa-gated in 1,585-cm2 roller bottle cultures of MA104 cells as described previously (25), usingculture mediumcontaining 25 pLgoftrypsin (Hazleton Biologics Inc., Lenexa, Kans.) per ml. For the preparation of virus which contained the uncleaved form of VP4, this medium was removed 6 h

postinfection and the monolayerswereextensivelywashed andreincubated with mediumlacking trypsinandcontaining 1%fetal calfserum. Theinfected cells were then harvested 40 h postinfection. Double-shelled virus was purified by

banding on CsCl gradients that contained 10mM Tris HCl (pH 7.5) and 10 mM CaCl2 (25) and stored at 4°C in CsCl priorto use.

Trypsin cleavage ofVP4 was achieved by first dialyzing thevirusagainstTBS (140mM NaCl,0.7 mM Na2HPO4,5 mM KCI, 20mMTris HCl [pH 7.5]), followed bydigestion

with1-,ug/mltolylsulfonylphenylalanylchloromethyl ketone (TPCK)-treated trypsin (Sigma type XIII) for 30 minat37°C.

Aprotinin (100 ,uglml; Serva GmbH & Co., Heidelberg,

Germany)wasaddedtoinhibitfurthertrypsindigestion,the viruswasrebandedon apreformed CsCl gradient, and then 4334

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CsCl was removed by dialysis against TBS. Samples were prepared for microscopy at concentrations of about 2 mg of viral protein per ml.

NH40H treatment of virus. Virus at 0.5mg/ml in 4 M CsCl was dialyzed against a solution containing 25 mM NaCl, 1 mM Tris HCl (pH 7.5), and 1 mM CaCl2. Aprotinin was added to a final concentration of 10,ug/ml. NH40H (1.25 M) (prepared from a 25% NH3 solution [analytical grade]; BDH, Poole, England) was added to the virus preparation to give a final concentration of 250 mMNH40H, and thepreparation was incubated at room temperature for 25 min. The treated virus (100,ul)was then overlaid onto a 20-,ul cushion of 30% sucrose prepared in TBS and contained in an airfuge tube (5 by 20 mm; Beckman Instruments, Inc., Palo Alto, Calif.). The mixture was then centrifuged at 22 lb/in2 for 90 s in a Beckman Airfuge with an A-100/30 rotor to pellet thesmooth particles which were resuspended in TBS for electron mi-croscopy. For protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (14), the supernatant (80pul) was first neutralized with acetic acid, and the protein was concentrated by precipitation with 9 vol-umes of ethanol at -20°Cfor 1 h.

Electron microscopy. Specimens were examined by using the low-dose facility of a Philips CM12 electronmicroscope operating at 80 kV. Suitable areas were recorded onKodak SO-163 film at a magnification of x28,000 and a dose of about 10 electrons per 0.01 nm2. The dose was calibrated against the speed of the film, which was developed in full-strength D19 for 12 min. The magnification was verified with catalase crystal spacings and maintained by using a constant (eucentric) objective lens current.

Cryoelectron microscopy. Films with perforations were made by dipping cleaned microscope slides into a 0.5% solution of collodion in acetone. The dried films were examined by phase-contrast microscopy to assess size and distribution of holes. Suitable films were floated ontowater, and 400-mesh copper grids were applied to the surface. These grids were picked up, coated with platinum-carbon (2.0 nm) toimprove electrical conductivity, and then coated with carbon (10.0 nm) using afreeze-fracture coating source. The plastic layer was dissolved away by rinsing individual grids in amyl acetate, and after drying the grids were examined once more. The very high contrast of theplatinum layer made it possible to identify false holes or bounded depressions. A

5-pI

sample of virus was applied to thegrid,

which was then blotted andplunged into liquid ethane (1). Without warming, the grid wastransferred toliquidnitrogen

and inserted into a cold stage(Gatan Inc.,Warrendale, Pa.) designed to maintain the specimen in amorphousice (5). To minimize contamination with ice in the microscope, a blade-type anticontaminator (10) was used in addition to the standard cold trap.

Negative staining. Optimal results were obtained using freshly prepared 10% uranylformate. One-milliliteraliquots of 1% uranyl acetate weredispensed into Eppendorf tubes. Then 0.1 ml of 1.0 M NaOH was added,and thetubes were sealed and shaken. The precipitate was sedimented by centrifugation, and the supernatant was discarded. The pellet was dissolved in 100 p1 of5% formic acid byvigorous

mixing and used immediately. A carbon-coated grid was floated on 5

p,l

ofvirus on a Parafilmsurface (AmericanCan Co., Greenwich, Conn.). After about 1 min, the grid was sequentially washed and stained bytransfer across 3

50-pd

drops of TBS and 2 drops of stain before itwasblotteddry

with filter paper held to the side of the grid.

Hemagglutination assays. Virus samples in TBS were

diluted in microtitration dishes

(Behring

Institute,

Behring-werke, Marburg, Germany),

yielding

diluted

samples

of

100-,ulvolume. Anequalvolumeofa

freshly

prepared

0.5% suspension of human group 0

erythrocytes

(suspended

in thesame

buffer)

wasaddedtoeach

well,

and the assaywas

read after 3 hat roomtemperature.

RESULTS

Rotavirus spike morphology.

Images

of rotavirus in ice show low-contrast

spikes

extending

from the surface of the intact virions (Fig. la) as described

previously

(23, 27).

Low-dose uranyl formate-stained

images

(Fig.

lb)

also re-veal these structures, which were radiation sensitive and whichwerelost

using

conventional

imaging

methods.

Figure

lb reveals that different numbers of

spikes

project

as a coronafrom individual

particles.

Thenumberof

spikes

isso low for some virions that the variation cannot be due to chance orientation but rather must be due to a reduced occupancy of the 60 sites in the

capsid.

Anotherfeature of the negatively stained

spikes

is the presence ofapparent Y andlooped conformations

(indicated

by

arrowheads in

Fig.

lb). However, while

initially

thiscould be takenas

support-ive evidence that the

spike

is

dimeric,

this may be due to

imagesof

neighboring

spikes

being

superimposed.

Virus that had not been

exposed

to

trypsin

showed the same wide

variability in numbers of

spikes

(Fig. 2a),

and these were more

frequently

seen as

single

straight

structures. The absenceofYand

looped

conformations in

undigested

prep-arationscould be

interpreted

as

indicating

the

dependence

of these structures on

prior

trypsin

treatment, but it

might

merely be the consequence of

variability

in

staining

condi-tionsbetween

preparations.

Removal of spikes by treatment at elevated

pH.

A wide rangeof

experimental

conditions were

investigated

for their

abilityto remove

spikes

from the intact virion as observed

bylow-dose electron

microscopy

and

gel

electrophoresis.

As

anticipated,

the

spikes

wereresistant to

sonication,

isopyc-nic

banding

onCsCl

gradients,

and other methods

tradition-allyused invirus

purification.

Digestion

withawide rangeof

proteolytic

enzymes,

including

chymotrypsin,

thermolysin,

papain,

trypsin,

subtilisin,

pepsin,

and V8 protease, was foundtobeineffective.Treatmentwith heat and exposureto urea, methods which

successfully

release reovirus

spikes

(8), also were ineffectual. The nonionic

detergents

Triton

X-100, Nonidet

P-40,

and

,-octyl

glucoside

also failed to

disrupt orsolubilize theviral

proteins.

While

investigating

the effects of

pH,

the best method which

reproducibly

removed the

spikes

yet

yielded

other-wise intact

particles

was found to be treatment with dilute ammonium

hydroxide.

Higher

concentrations of this weak base were found to release an

increasing

proportion

of

spikes, withaconcentration of 250 mM

being

optimal (Fig.

2b) atpH 11.2. Factors other than

pH

must be involved in this processas

NaOH-phosphate

and

NaOH-glycine

buffers

causedreleaseataround

pH

13 butalso

disrupted

thevirus. The material in the

background

of

Fig.

2b is taken to be released

spike

protein.

When

separated

by

centrifugation,

the

resuspended

pellet

brought

to

pH

7.4

(Fig.

2c)

gave

images of intact

particles

with a

good

circular outline and evidence for

good

structural

integrity.

Inthese

viruses,

itcan

be seen that the stain-filled channels

running through

the viral coat are more

highly

contrasted where the

spikes

are

absent.

The supernatantderived from the

NH40H

treatment

(Fig.

2d) was free of intact

particles

and viral capsomeres but

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~~

A

FIG. 1. Rotavirus(isolatedin thepresence oftrypsin) imaged by cryoelectron microscopy inamorphous iceandby low-dosenegative stainingwithuranyl formate.(a) Animage ofthe unstainedpreparation,underfocusedby3,um, shows therandomly orientedintactparticles in the thinlayer (about100 nmthick) of ice. Spikescanbe seenprojectingfrom thesurfacewithanindication ofaterminal domain.(b)In

astainedpreparation imagedcloser tofocus,theprojectionsexhibitloopedand bilobed conformations(indicated by arrowheads)but these maybethe resultof overlap betweenpairs or groups of spikes. Bar, 100 nm.

contained material exhibiting globular and short fibrous structures. Althoughthis material could not be identified by microscopy, SDS-PAGE (Fig. 3) showed that the released material (lanes4and8) wasalmostexclusivelyVP4(orVP5* and VP8* in trypsin-treated virus). The spike protein was almost completely released by alkaline treatment while the major external protein VP7wasretained (Fig. 3, lanes 3 and 7). Minor amounts of VP6 present on the gel must derive from a small fraction of particles that are either disrupted

during centrifugation or inefficiently sedimented, because this proteinwasalso present in the controls (Fig. 3, lanes 2 and 6).

To confirm that the proteins of the virus have not been denatured by the ammonium hydroxide treatment, the

treatedparticleswereincubated withamonoclonalantibody

that recognizes the serotype-specific epitope present on

region A of the single large immunodominant neutralizing

domain of VP7 (18). Cryoelectron microscopy (Fig. 4) re-vealed that the monoclonal antibody bound to the smooth

particle, implying that alkaline treatment had not

grossly

denatured theVP7proteinwhich remainedonthesurface of thesmoothparticle.

Hemagglutination.Inview of theassignmentof VP4asthe viralhemagglutinin(11), thehemagglutinatingability (HA)of virus treatedtoeither cleaveorremoveVP4wasdetermined by using human erythrocytes. Table 1 shows that virus

prepared in the presence oftrypsinexhibited aconsistently

lowerhemagglutinationtiter(approximatelytwo- tofourfold

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FIG. 2. Effect of ammonium hydroxide treatment on rotavirus as revealed by negative staining and low-dose microscopy. (a) The uncleaved (VP4) preparationof virus (cf. with Fig. lb) shows manysingle spikeprojections. (b) Virus preparation followingadditionof ammonium hydroxide and incubation at pH 11.2. Note the complete release ofthe spikes from the virus surface. The material in the backgroundmay be releasedspikeproteinwhich appearsaggregatedwhenstainedatthishighpH. (c) Particles separated bycentrifugation and brought to neutral pH have a smooth circularprofile and show very clear stain-filled channels radiating through the capsid. The background is free of released protein. (d)ThesupernatantbroughttoneutralpHyields images(white arrowheads) ofshort(20-to40-nm) linear moleculeswhich may be the releasedspike protein. Small globular material may be either denaturedprotein fromthespikeorthe inner capsidVP6. Bar, 100nm.

0, fc.

b

'41..

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Trypsin

pH

11.2 + +

P S P S P S PS

1 1 1 A C A< 7

VPI -VP2,3 -VP4f VP5* -VP6 VP7 -VP8* -20

FIG. 3. The spikeis composedofpolypeptideVP4orits cleav-age productsVP5* and VP8*. 12.5% SDS-PAGEanalysisof rota-viruspolypeptidesderived from virusbearingintact VP4(lanes1 to

4)ortrypsin-cleaved VP4 (VP5* andVP8*, lanes 5 to 8). Control virus (lanes 1, 2, 5, and6) was centrifugedto yield pellet(P) and

supernatant(S)fractions. Pellets andsupernatants generatedby pH

11.2treatmentareinlanes 3 and 4(uncleavedvirus)andlanes7 and

8(cleaved virus).Lane9 contains molecularmassmarkers(sizesin

kilodaltons). LargearrowsindicateVP4;smallarrowsindicate VP5*

and(faint)VP8* bands.

less for an equivalent number ofparticles) than virus

pre-pared inthe absence oftrypsin (preparation 1 versus 2 and

preparation 3 versus 4). Preparation 5,whichwas grown in the absence oftrypsin but then subsequently digested with

trypsin, also showed a reductionin HAtiter. Thus, trypsin cleavage consistently yielded virus that exhibited a lower HAtiter. Followingalkaline treatmenttoremoveVP4,both cleaved and uncleaved viruslost all detectable HA and no

HAcould be recovered from thesupernatant.

DISCUSSION

In their study of tobacco mosaic virus, Williams and

Fisher(26) appliedlow-dose methods toreveal finedetail of negativelystainedpreparations. Cryoelectronmicroscopyis

dependentonthesetechniquesbecauseof thehighradiation sensitivity of unstained biological molecules in ice. The

contrast in suchan imageismuch lower thanthatproduced by stained specimens, but the preparation is free of the compressionanddistortion causedbydrying. Consequently, computational averaging methods are used to overcome

noise and generate athree-dimensionalmodel. Prasad et al. (23) and Yeager et al. (27) applied icosahedral averaging

methods to images of rotavirus produced by cryoelectron microscopyandderived the structureof theaveraged spike.

Their work revealed the spike to have a complex bilobed

morphologyandFabdecoration led to theinferencethat the spike is adimerof VP4(22).

Ourfindingsshowthat thesurfaceprojectionsof rotavirus canbe imagedin stainbylow-dose methods. In the proteo-lyzed state, the Y andlooped conformations observed sug-gestthe opening up ofa dimer, in agreement with the Fab

decoration data (22). The spike images, however, could be superimpositions, because inthe polarprojection,

contribu-tions totheimagemaybe madefromboth aboveandbelow theequator. A full interpretationof the structuresobserved

would be dependenton thegeneration ofacomplete three-dimensional model; such ananalysis isbeyondthe scope of this report and in anyeventcould bemisleadingbecause of the distorting effects created by specimen

dehydration.

Neithercanpossible variations in conditionsduringstaining

be excludedas apossible explanationforourfindingthat the spike is in a straight conformation when unproteolyzed.

However, ourresults dosuggest thatarigorous analysis of the effects oftrypsinon spike morphology should be under-takenby cryoelectron microscopyandimage analysis.

By specifically releasing the spikes from the virus, we have shown that the constituent protein is VP4 (VP5* and VP8* aftertrypsin treatment). This observation agrees with the results of Fab decoration(22) which also localized VP4 tothespike. We have also shown that VP7 doesnotform a part of thespikeand thatfollowingreleaseofVP4,the

major

neutralizing epitope of VP7 is correctly presented on the surface of the virus. Consequently, it is reasonable to conclude that the presence of VP4 is not necessary fora structurally sound outershell, indicatingthatVP4 and VP7 probably do not formjoint domains on the surface of the virus. The lack of expected stoichiometry found between these proteins (15) and the frequently observed "bald" regions in virus images (both in ice and in stain) could be explained in the following two ways. (i) VP4, which lacks a signal sequence (16) and is cytoplasmic, associates rather randomly with the virus as it buds through the membrane (20, 21). (ii) Some of the spikes could be lost during isolation of the virus. Since the virus does not encounterahighpH

environmentduring the purification proto ol and the spikes

on the virus areotherwise very refractory to extraction, loss ofspikes during purification appears unlikely.

The closely related reovirus also has surface projections

and these have been shown to be composed of a single

protein, sigma 1 (8). There is no sequence homology be-tween VP4 and sigma 1, and there is no evidence for a coiled-coil alpha-helical domain in VP4. However, as both theseproteinsareviralhemagglutinins, the appearanceofan apical globular domain on VP4 may be linked with this activity. The fact that reduced HA was found for trypsin-treated rotavirus is enigmatic but confirms the observations of Kitaoka et al. (13); trypsin treatment is known to enhance infectivity (4, 6, 7) and might be expected to increase, rather than reduce, the interaction of virus with cell membranes. We have shown(Fig. 3) that trypsin does not release either of the two cleavage products (VP5* and VP8*) from the virus. Thechange in HA may be linked to a possible change inshape of the spike. Two reconstructions of rotavirus have shown spikes with bilobed distal domains. Yeageretal. (27) point out the effect low occupancy of spikes on the virus surface would have on the averaged model. In light ofour findings, we believe that it is important to determine the extentofproteolysis in preparations studied by cryoelectron

microscopy so that any structural alterations which occur

following cleavage can be defined.

Finally, this work has demonstrated that the smooth

(spikeless) particles which lack VP4 retain their structural integrity. They havean undistortedcircularprofile andgive every expectation of being suitable candidates for image reconstructionusing cryoelectron microscopy (2a). By

com-parisonwith nativevirus,itshould bepossible to determine thestructureof theanchoringpartofVP4within the capsid. Further investigation of the smooth particle therefore may

helpto unraveltheinteractions of the inner and outershell

proteins of rotavirus and the structural features that sur-round the base of the spike.

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FIG. 4. Smooth particles retain the epitopefor theVP7-specificmonoclonalantibody 159. (a) Smooth particles preparedasforFig. 2c but unstained inamorphous ice andunderfocused by3,um.(b)Smoothparticles preparedasin panelabutdecoratedwith monoclonal antibody 159(18) by mixing thealkali-treated virus with antibody and incubatingatroomtemperaturefor2 hpriortofreezing. Bar, 100nm.

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TABLE 1. Hemagglutinationby rotavirus particlesfollowing trypsin cleavage or pH11.2treatment

Virus Cleavage Alkali HAtiter

prepn' stateb treatment (U/mg ofvirus)

1 - - 11,428

2 + - 5,818

3 - - 22,068

4 + - 7,655

5 - - 22,068

+ - 11,034

6 + - 27,526

+ + <100

7 - - 102,400

+ <200

a Seven individuallypurified viruspreparationsyielded differentHAtiters.

bCleavage of>98% (+) and <10% (-), as determined by SDS-PAGE

analysis.

ACKNOWLEDGMENTS

This workwas supported bygrants fromthe Medical Research Council ofNewZealand and theNew ZealandChildren's Health ResearchFoundation.J.A.B. was a NewZealandUniversity Grants Committee Research Fellow.

Wethank AlasdairSteven forhelpful criticismof themanuscript. HarryGreenberggenerouslysuppliedtheVP7-specific monoclonal antibody159 andprovidedhelpful suggestions forthepreparation of uncleaved virus.WethankBarryBenning fortheconstruction ofa supplementary coldtrapfor the electronmicroscope.

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