Structural proteins of La Crosse virus.

JOURNAL OF VIROLOGY, Sept. 1976,p.985-997 Vol. 19,No.3

CopyrightC1976 American Society for Microbiology Printed in U.S.A.

Structural Proteins of La Crosse Virus

JOHN F. OBIJESKI,* DAVID H. L. BISHOP, FREDERICK A. MURPHY, AND ERSKINE L. PALMER

Bureau ofLaboratories, Center for Disease Control, Atlanta, Georgia 30333; and Department of Microbiology, University of Alabama, Birmingham, Alabama 35294*

Received for publication 19April 1976

Preparations of La Crosse virus, a member of the California encephalitis groupofbunyaviruses,werefound to possess three major virion proteins. Twoof the proteins were glycosylated (Gi and G2) and were located on the surface of thevirusparticles. These twoglycoproteinswerepresent inequimolaramounts and possessed apparent molecular weights of 120 x 103 and 34 x 103. Virion

nucleocapsids, isolated by a nonionic detergent and salt treatment, contained

another major protein, N (molecular weight = 23 x 103). A large, but minor,

protein species L (molecular weight = 180 x 103) was also found in virus

preparations. Theapproximatenumber of protein moleculesper virion has been

determined. Electronmicroscopy of purified LaCrossevirusindicated that the

virus particle (mean diameter, 91 nm) is enveloped and possesses irregular

surface projections (length, 10 nm).

LaCrosse viruswasinitially isolatedin 1964

from frozenbrain tissue of a 4-year-old female

child whodiedin1960from

meningoencephali-tis in La Crosse, Wis. (29). Comparative

neu-tralization, complement fixation, and

immuno-diffusion tests have shown thatLaCrosse virus

is serologically related to, but distinguishable

from, other isolates of the California encephali-tisgroupofbunyaviruses (3, 12-14, 22, 24, 31).

Thevirus has been isolated from various

mos-quito speciesthroughout the north-central and

eastern United States, and neutralizing

anti-bodies have been detected in various

forest-dwelling

small mammals inthese same areas

(11, 28). Since 1964, several hundred cases of

encephalitis have been attributed to the

Cali-fornia encephalitis group of viruses,

particu-larly inOhio, the southwestern region of

Wis-consin, and othermidwestern states (5).

Apart fromaninitialstudy by McLerran and

Arlinghaus (9),relatively littleisknown about

the chemical composition and structure of La

Crosse virus. We have investigated the

num-ber,

location,

and function of theproteinsofLa

Crosse virus and characterized the purified

vi-rusparticle byelectron microscopy. MATERIALS AND METHODS

Reagents. Uniformly labeled L-14C- or 3H-amino acid mixtures, [3H]uridine (32 Ci/mmol),

D-[3H]glucosamine (10 Ci/mmol), D_['4C]glucosamine (0.2Ci/mmol), [32P]orthophosphate, and ['25I]iodine (17 Ci/mg) were obtained from New England Nu-clearCorp., Boston, Mass. Phosphorylase A, a-ga-lactosidase, and pancreatic RNase were purchased from Worthington Biochemicals Corp., Freehold,

N.J. Cytochrome c, myoglobin, chymotrypsinogen, ovalbumin, and bovine serum albumin were ob-tained from Mann Research Laboratories, New York, N.Y.; bromelain and the purified grade of lactoperoxidase were purchased from Calbiochem, La Jolla, Calif.

Growth and purification of viruses. Vesicular stomatitis virus (VSV), Indiana serotype, was grownand purified as described previously (15). La Crosse viruswasobtained from D.Trent, Center for Disease Control, Fort Collins, Colo., and was fur-ther plaque purified in confluent monolayers of BHK-21cells. To prepare working virus stocks, we infected BHK-21 cellsinpetriplates with <10 PFU ofvirus,andat 48hthe virus presentin oneplaque plug was usedtoinfect107confluent BHK-21 cells. This culture was harvested at 48 h postinfection and storedinaliquotsat -70°Cin amedium containing 10%fetal calfserum.Thetiterof virusinthe stock preparationwas 8 x 107PFU/ml.

Roller bottlecultures of confluent BHK-21 cells (1 x 101 to 3 x 10# cells/bottle), grown in reinforced Eagle medium supplemented with 10% fetal calf serum, were infected with the stock virus at an inputmultiplicity of infection (MOI) of0.001 PFU/ cell. Fortheproduction of labeled virus,weincluded in the growth medium either L-'4C-labeled amino acids (0.2 uCi/ml), D_[4C]glucosamine(0.2/,Ci/ml), L_3H-labeled aminoacids (2 ,uCi/ml), D_[3H]glucosa-mine (2

ACi/ml),

[3H]uridine (5

tLCi/ml),

or [32P]_ orthophosphate (100 uCi/ml). When labeled amino acids were used, the concentration of unlabeled amino acidsinthegrowth mediumwasreducedto 20%of the normal level.

After 48 h ofgrowth at 33°C, the infected cell culture fluids wereclarified by low-speed centrifu-gationat10,000 x gfor 20 min in aBeckman J-21 centrifuge to remove cell debris. The subsequent purification stepswere essentially those described 985

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986 OBIJESKI ET AL.

previously for the purification of other enveloped viruses (1, 15), with certain modifications to im-prove the yield of intact virions and reduce the amountof contaminationby cellular material in the final virus preparation. Virus wasprecipitated from the clarified culture fluids by adding polyethylene glycol 6000 (Carbowax 6000, 70 g/liter)and NaCl (23 g/liter). After the mixture wasstirred 4 h at 4°C, the virusprecipitate was collected by centrifugation at 10,000 x g for 20min, resuspended in 0.01 M Tris-hydrochloride buffer, pH 7.6, containing 0.15 M NaCl and 0.003 MEDTA (TSEbuffer), clarifiedby centrifugation, and loaded over combination equi-librium-viscosity gradients of glycerol and potas-sium tartrate (KT-GLY) (15). After the gradients werecentrifuged 3 h at 4°C (40,000 rpm in a Spinco SW41 rotor), thevisible virusband wascollected by pipette anddialyzed overnight at4°C against TSE buffer. The virus was thenloaded onto a gradient of 20 to 70% (wt/vol)sucrose in 1 M NaCl, 0.01 M Tris-hydrochloride buffer, 0.002 M EDTA, pH 7.4, and centrifuged at 4°C and 35,000 rpm for 3 h. The virus band was harvested, diluted fourfold with TSE buffer, andpelleted through a 1-ml cushion of 30% (wt/vol) sucroseinTSEbuffer by centrifugation for3 hat 35,000 rpm. The final virus pellet was resus-pendedin 0.5 to1.0ml ofTSE buffer and storedat 4°C. Virus protein concentrations were usually be-tween 0.2and 1mg/ml.

Protein determinations and polyacrylamide gel electrophoresis. Protein concentrations were deter-mined by the method of Lowry et al. (7), with bovine serum albumin as aproteinstandard. The separa-tion of sodium dodecyl sulfate (SDS)-dissociated whole virion proteins by electrophoresis hasalready been described (15), using either an 8% (wt/vol) continuous polyacrylamide gel involving a phos-phatebuffer, pH 7.0, and SDS(cont-SDS);a 3 to30% (wt/vol) lineargradientpolyacrylamidegel and the same buffer-SDS system (gradient-SDS); or a dis-continuous gel system involving a 3.6% (wt/vol) stacking gel and a10%(wt/vol)resolving polyacryl-amidegel plus SDS andadiscontinuous buffer sys-tem involving Tris-hydrochloride and glycine buffers,pH 9.0(disc-SDS).Theseparation obtained forlabeled protein specieswasdeterminedbyslicing frozen gels into 1-mm portions and determing their content ofradioactivity after soaking the slices for 18 h in a toluene-based scintillation cocktail con-taining 3% (vol/vol) NCS (Amersham-Searle, Chi-cago, Ill.). The separation ofprotein species was determinedinsomecases by staininggels with Coo-massie brilliantblue, destaining in an acetic acid-methanol mixture, and scanning the gels at 640 nm, asdescribedpreviously (1).

Iodination of viral proteins. Purifiedvirus prepa-rations containing 200

gg

ofproteinin 0.4 mlof0.1 Msodiumphosphate buffer, pH 7.0, wereincubated at20°Cwith 250,uCi of carrier-free['251]iodinein the presence of10,ugoflactoperoxidase and0.01 ml of freshly diluted 0.001 M H202 (26). Five additional 0.01-mlportions ofhydrogenperoxidewereaddedat 5-minintervals (33). After the lastaddition, 2mlof cold (4°C)TSE buffer and0.1ml ofsaturated potas-sium iodide solution were added. Unreacted

re-agents were removed from the virus particles by loading the reactionmixture on a20to70%(wt/vol) gradient of sucrose containing 1 M NaCl, 0.01 M Tris-hydrochloride buffer, 0.002 M EDTA, pH 7.6, and centrifuging at 35,000 rpm for 1 hat4°C. The visible virusband wascollected with a pipette and dialyzedagainst TSE buffer. The specific activity of thelabeled virus preparation wasdetermined to be ontheorder of 105 cpm per mg of protein.

RESULTS

Growth curves of La Crosse virus as a func-tion of the input MOI. Confluent monolayers

of3 x 106 BHK-21 cells in 25-cm2 flasks were infected with virus at input MOIs equivalent to 10,

1,

or 0.01 PFU/cell. The monolayers were incubated at 33°C; the release of infectious vi-rus wasmeasured bydetermining the number of PFU presentin thesupernatant fluids. Ina separate experiment, the virus present in a

single plaque plug was used to infect a

con-fluent monolayer of3 x 106BHK-21 cellsat an

inputMOI of0.002 PFU/cell, and the yield of

infectious virus was also determined (Fig. 1).

Thehighestvirusyield wasobtained from the

cellsinfectedatthe lowest

MOI,

but was

sup-_-o----o.002PFU/CELL

/ ~~~CLONED

PLAQUE

/ 0

0 .01 PFU/CELL

/0

lo7/ -' -A PFU/CELL

0

Qo0

;,r_

i,

PFU/CELL

I / *v

II

0~~~~~~~-I-I.-1--I,!

0AII

12 1/

12 24 36 48 60 72

HOURS POST INFECTION

FIG. 1. Growth curves ofLa Crosse virus as a

function ofthe input multiplicity of infection. Mono-layer culturesofBHK-21 cells, infectedwith 0.002, 0.01, 1, or10PFUlcell, weremonitoredforthe pro-duction of infectious virus by aplaque assay

tech-nique.

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LA CROSSE VIRUS 987

pressed by even moderate increases of MOI

(Fig. 1). At the calculated input MOI of 0.002

PFU/cell, about 150 infectious units were

pro-ducedpercell. By 48 h postinfection, there were

severe cytopathic changes in all cells and no

further virus production. In light of these

ob-servations, all further virus preparations were

prepared from cultures infected at an MOI of 0.001 PFU/cell.

Electron microscopy of purified virus

prep-arations. A low-magnification electron

micro-graph of gradient-purified La Crosse virus is

presented in Fig. 2A. Large numbers of virus

particles with a mean diameter of91 nm were

observed, and therewas noevidence of

contam-inating cellular debris. Fine, irregular surface

projections, 10 nm in length, were present on

the surface ofall particles (Fig. 2B). No

sym-metric arrayof theseprojections wasobserved,

in contrast to the results described for

Uuku-niemi virus (2, 18, 21, 23). Where negative-contrast medium penetrated the particles, the

envelopecould beseen, andinsomeparticlesa

convoluted, internal structure,

possibly

repre-senting theviralnucleocapsid(s), wasobserved

(Fig. 2C).

Sedimentation coefficient of La Crosse

vi-rus. Purified test samples ofLa Crosse virus

(1.5 mg/ml) were dialyzed against a reference

buffer (0.01 M borate, 0.16 M NaCl, pH 8.0),

and thesedimentation

coefficients

at20°Cwere

determined atspeeds of10, 12, 14, and 17,000

rpm in aBeckman modelE ultracentrifuge

op-erated with Schlieren optics. Sedimentation

coefficients (sapp) werecorrectedto

s,,,8w

values

by a standard procedure (27). By this

tech-nique, LaCrossevirus wasdeterminedtohave

asedimentation coefficient of 415S.

Identification and characterization of the

structural proteins of La Crosse virus. The

following procedure was used to identify the

structural proteins of La Crosse virus and to

estimate their molecular weights. Asample of

:'H-amino

acid-labeled La Crosse virus was

mixed with a sample of

'4C-amino

acid-labeled

VSV Indiana virus, and their proteins, were

dissociated by SDS. The VSV Indiana

struc-tural polypeptides were included as markers

becausethey are known to haveapparent

mo-lecularweights of 160x 10: (L),65 x

10:3

(G), 54

x 103 (N), 42 x 10:3 (NS), and 27 x 103 (M) in

cont-SDS-gels (30). The proteins of the two

vi-ruses weresubjected to electrophoresis in

poly-acrylamidegels containing SDS. Three gel

sys-tems, cont-SDS, disc-SDS, and gradient-SDS

(15; Espositoand Obijeski, inpreparation),

de-scribed in Materials and Methods, were used. These particular gelsystems were used because

proteinsof similarmolecularweightscanoften

be separatedby onegel system but notby

an-other(16,25). Also,anestimation of the

appar-entmolecularweightsofthe VSV Indiana

pro-teins, even in the presence of SDS, depends

uponthegel systemused (15).The

electropher-ograms of the proteins of the two viruses are

presented inFig. 3.

The molecularweights of the structural

pro-teinsof La Crossevirus, asdetermined by com-parison with the known major VSV Indiana structural proteins and seven standard

pro-teins, are shown in Table 1. Four structural

polypeptides were identified in the

cont-SDS-gel system, alarge protein (180 x 103daltons,

minorcomponent), a majorpolypeptide (120 x

103 daltons), a third protein present in

appar-ently smalleramounts(34 x 103daltons), anda

fourthcomponent(23 x 103daltons). No

signifi-cant differences in the apparent molecular

weightsor in the number of viral protein

spe-cies were observed by using the other two gel

systems.

Identification oftwovirusglycoproteins in

LaCrosse virus. La Crossevirus wasgrown in

the presence of '4C-labeled amino acids and

[3H]glucosamine,

purified,

and dissociatedwith

SDS and mercaptoethanol. The

differentially

labeledvirusproteins wereresolvedby

electro-phoresis in 8% polyacrylamide gels (Fig. 4A).

The largest and smallest viral proteins were

labeled only by the '4C isotope. However, the

twointermediate-sizedproteins werelabeled by

bothisotopes (3Hand 14C), thereby identifying

them as glycoproteins; these proteins were

designated Gl and G2.

Demonstration that the glycoproteins are

atthesurface ofthevirus

particle.

Apurified

virus preparation labeled by '4C-amino acids

and [3H]glucosamine wasincubated with 1mg

of the proteolytic enzymebromelain per ml in

TSE buffer for 2 h at 35°C to remove the

pro-teins located on the outersurface ofthe virus

particle (10). The bromelain-treated virions

were reisolated bycentrifugation in agradient

of sucrose anddissociated by SDS. After

poly-acrylamide gel

electrophoresis,

it was found

that neither ofthe viral glycoproteins (Gl or

G2) had survived the proteolyticenzyme

treat-ment (Fig. 4B). The other two viral proteins remained associated with the protease-treated virions. Thebromelain-treated preparation ex-amined byelectron microscopy contained only

spikeless particles with intact envelopes (Fig.

5). The infectivity ofspikeless particles as de-terminedbyplaquinginBHK-21cell

monolay-ers was about 10- that of untreated control virus preparations (data not shown). It was VOL. 19, 1976

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FIG. 2. Electron micrographs ofpurified La Crosse virus, all negatively stained with 2% sodium phospho-tungstate. (A)Concentratedsuspension ofpurified LaCrosse produced by thedescribedprotocol ( x62,400). (B) Virusparticles showing the characteristic layer of irregular surface projections (x201,500). (C) Virus particles penetrated by the negative-contrast medium showing an electron-lucent membraneand internal structure(x152,400).

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LA CROSSE VIRUS 989

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FRACTION NUMBER

FIG. 3. Electropherograms of VSVIndiana and La Crosse viralproteins. Mixtures of'4C-amino acid-labeled VSV and 3H-amino acid-labeled La Crosse viruspreparations were dissociated by SDS and

2-mercaptoethanol,andthe proteinswereresolvedbyelectrophoresisincont-SDS-,disc-SDS-,

orgradient-SDS-gels (see text).After electrophoresis eachgel wassliced into 1-mm segments,and the distribution ofeach

isotopeinthegel wasdetermined byliquid scintillationspectrometry.

TABLE 1. Molecularweights(xlO-3) of La Crosse virus proteins"

Cont-SDS Disc-SDS Gradient-SDS

VSVIndiana LaCrosseb VSV Indiana LaCrosse VSV Indiana La Crosse

L 160 L 180 L 149 L 190 L 160 L 175

G 65 G1 120 G 63 G1 125 G 63 G1 120

N 54 G2 34 N 46 G2 38 N 53 G2 37

NS 42 N 23 NS 54 N 25 NS 42 N 22

M 27 M 27 M 27

aThe molecular weights of the various viral proteins were estimated by comparing theirelectrophoretic mobilities relative to seven marker proteins runinthe same gel system(,8-galactosidase,phosphorylase A, bovineserumalbumin, ovalbumin,chymotrypsinogen, myoglobin, and cytochrome c). The values reported arethemeans offiveseparatedeterminations.

b Thedesignation of La Crosse virus proteins is based on the results giveninthetext. VOL. 19, 1976

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990 OBIJESKI ET AL.

12F

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FRACTION NUMBER

FIG. 4. Identification ofLa Crosse virus glycoproteins. (A) Purified La Crosse virus, grown in the presence of '4C-labeledaminoacids (a) andf:3H]glucosamine(0) was dissociated by SDS and 2-mercaptoethanol, and the proteins were resolved by cont-SDS-gel electrophoresis. (B) A similarly labeled virus preparation was treated with bromelain andrepurifiedby centrifugation in a 20 to70% gradient of sucrose containing TSE buffer and 1 MNaCl(35,000 rpm for 45min). The visible band ofparticles was recovered and dissociated by SDSinthe presence of 2-mercaptoethanol; the proteins were resolved by cont-SDS-gel electrophoresis. concluded from these results that the

glycopro-teins constitute thespikesonintact virus

parti-cles.

Additional studiesweredonetoconfirm that theglycoproteins weretheonlyvirion proteins

externaltothe viralenvelope.Apreparationof unlabeled viruswasiodinatedwith '25']iodine, lactoperoxidase, and hydrogen peroxide. After repurification by equilibrium gradient centrifu-gation, the labeled virus particles were

re-covered. An aliquot was dissociated by SDS, and theproteinswereresolved by

electrophore-sis in an 8% cont-SDS-gel. Although some

la-beled material remained at the surface of the

gel,

the majority of the label

migrated

with

electrophoretic mobilities characteristic of the Gl and G2 glycoproteins (Fig. 6). A small

amount of labeled material was recovered around fraction 40. No label was observed in the regionsofthe gel where thetwo

nonglyco-sylated viral proteins were expected. Another

aliquotof the iodinatedvirusparticleswas

in-cubated with bromelain and repurified by

su-crose gradient centrifugation, and the visible

band ofparticleswasrecovered. After

dissocia-tion of the particles by SDS and resolution of the constituent proteins by electrophoresis in

an 8% polyacrylamide gel, it was found that

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LA CROSSE VIRUS 991

almost all the label

originally

associated with

thetwoviralglycoproteins (aswellasthe

frac-tion 40 material) had beenremoved by the en-zyme treatment (Fig. 6).Iodinationof Triton

X-100-disrupted particles, followed by

trichloroa-cetic acid precipitation and dissociation with

SDS, labeled all four structural proteins (data

notshown).

Gradient

centrifugation

of

bromelain-treated La

Crosse

virus.When La Crosse virus

was incubated with

bromelain,

the

density

of

thespikeless particles was 1.16

g/ml

in

combi-nation KT-GLY

gradients

(Fig. 7). Control

preparations ofvirusincubated intheabsence ofenzyme had a

density

of 1.20

g/ml.

When

virus preparationsradiolabeled withamixture

ofaminoacidsweretreatedwithbromelain and

centrifuged in KT-GLY

gradients,

approxi-mately40%of thetotal

radioactivity

wasfound

to be associated with the

1.16-g/ml region,

whereas theremainder of the

radioactivity

was

atthetopof the

gradient (Fig.

7C).

Incontrast,

when

glucosamine-labeled

viruswasincubated

with theenzyme, >90%of thetotal

radioactiv-ityremainednearthetopof the gradient(Fig.

7B). Similarly, when '2I-labeled virus was

in-cubated withbromelain, almost all of the radio-activity was released and remained near the origin ofthe gradient (Fig. 7A). Labeled mate-rial from the 1.16-g/ml region wasprecipitated

completely with trichloroacetic acid and could

be recentrifuged in KT-GLY gradients.

Fur-thermore, electron microscopy of the particles

from thisregionofthe gradient showed that the

particles were devoid of their surface

projec-tions (see Fig. 5). Less than 10%ofthe

brome-lain-solubilized radioactive material, which

re-mained at the top of the gradient, could be

precipitated withacid.

Solubilization of La Crosse glycoproteins.

Exposure of many lipid-containing RNA

vi-ruses to nonionic detergents and various salt

concentrationshasbeen showntodifferentially

solubilize viral proteins (4, 6, 8). Purified La

Crosse virus labeled with :H-amino acids

sus-pended in 0.01 M Tris buffer (pH 7.8) was

treated with Triton X-100 (2% vol/vol) and

varying amounts of NaCl. Each preparation

X,;

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::i

F"-.-.4.4 '. _'l.

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qu

l1'-.rps r

i-*Ar

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FIG. 5. Electronmicrographof bromelain-treatedLaCrossevirusparticles.Repurifiedbromelain-treated LaCrossevirus(Fig.4B)wasnegativelystained withsodiumphosphotungstate(x132,300).

VOL. 19, 1976

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992 OBIJESKI ET AL.

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20

40

60

80

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FRACTION NUMBER

FIG. 6. Iodination ofintactLa Crosse virions. Unlabeled La Crosse virus, iodinated and repurifiedas describedinthe text,wassolubilizedby SDS and 2-mercaptoethanol, and the proteinswereresolvedby cont-SDS-gel electrophoresis (a). After the iodinated viruswastreated with bromelain (see text), theparticleswere

repurified,dissociated by SDS, and again resolved bycont-SDS-gel electrophoresis(0).

wasseparated intoa soluble and aparticulate

fractionbycentrifugation, and theprotein

con-stituents in each fraction were determined

by

SDS-gel electrophoresis. Figure 8C indicates

that only 5 to 10% of the

largest

La Crosse

glycoproteins (Gl and G2)weresolubilized (Fig.

100 treatment alone. However, when the virus

was treated with either 0.5 M or 1.0 M NaCl

togetherwith TritonX-100, almost100%of both

glycoproteins (Gl and

G2)

wassolubilized

(Fig.

8A, B). The

particulate

fractions of the

salt-Triton X-100-treated viruscontained

virtually

all of the

largest

and smallest viral

proteins.

Identification of the proteins associated

with the nucleocapsids ofLa Crossevirus. In

preliminary attempts to release the viral

nu-cleocapsids

of La Crosse virus with nonionic

detergents, we found that

only

when virus

preparationsweretreated with TritonX-100 in

the presence of NaCl could

nucleocapsids

be obtained essentially free from the viral

glyco-proteins (see Fig. 8).

Apurified preparation of La Crosse virus, la-beled by L3H]uridine and '4C-amino

acids,

was

dissociated by 2% (vol/vol) Triton X-100 in the presence of 1 M NaCl, and the mixture was

loaded on a KT-GLY gradient containing 1 M NaCl. After 24 h of

centrifugation

at

4°C

and 40,000 rpmin aSpincoSW41 rotor,itwasfound

thatessentiallyall the :1H label waspresent in

asingleband (p = 1.32)

together

with

approxi-matelyone-thirdof the '4C label (Fig. 9A). The

rest of the '4C label was recovered as a broad band ata density of 1.20 g/ml. To identify the viral proteins associated with the viral RNA, material from both regions of the gradient was

dialyzed free from the glycerol and potassium

tartrate and precipitated by 5 volumes of 5% (wt/vol) trichloroacetic acid at 4°C. The precipi-tates were recovered and dissociated by SDS,

andthe proteins were resolved by electrophore-sis in 8% cont-SDS-gels. The two proteins asso-ciated withtheviral RNA (fraction I material) were the smallest and the largest species (Fig.

9B). Since the smallestprotein is a majorviral

constituent (based on the number of molecules per virion Lsee below]) and is associated with the viralRNA, it can be tentatively designated asthe majornucleocapsidprotein (N). Because of its size, the large protein, also associated with the nucleocapsid, is designated L until a

function can be ascribed to it. The fraction II

material was found to consist of the two viral

glycoproteins (Fig. 9C).

Number of protein molecules in La Crosse virus particles. The approximate number of the various viral proteins was determined based upon the following assumptions: (i) that the estimatesof the apparent molecular weights of the individual viral proteins given in Table 1 are correct; (ii) that virions contain only one molecule of each size class of viral RNA; and

(iii)that the combined gram molecular weight

of the viral genome is 5.1 x 10' (unpublished

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VOL. 19, 1976 LA CROSSE VIRUS 993

A

X

observation).

In

determining

thevirion ratio of

P

=1.20

RNAto

protein,

a

5-ml

suspension

of La Crosse

virus labeled by [3H]uridine containing 0.260

8

mg of protein perml, andatotal of1.56 x

10"

cpm was used. After extraction, the purified

RNA had a

specific

activity

of3.53 x 107 cpm

O 1- per mg of RNA. It was calculated, therefore,

that therewas 1.3mgof protein to 0.044mgof

1

l viral RNAinthe

original

virus

suspension,

i.e.,

a.. Ithat therewas a

protein-to-RNA

weightratioof

o e 30:1.

~- 4 l Another

sample

of LaCrosseviruswas

disso-C9

[ ciated by SDSand precipitatedwith 5% (wt/vol)

trichloroacetic acid. The

precipitate

was

re-covered bycentrifugation and washed by

alco-P4.1611

hol, andanaliquotwastreated with SDS before

^^A electrophoresis in an 8% (wt/vol)

polyacryl-amide gel. After electrophoresis, the gel was

stained

by

Coomassie brilliant blue and

scannedat 640 nm. Theareaunder eachpeak of

thescan wascomputed, and themassratio was

A 6 determined for the four viral proteins. From

10 three such

determinations,

the mean

propor-tions of virion proteinrepresented by the L, Gl,

G2, and N species were 3, 51, 14, and 32%,

ll0 e

respectively.

Similarresults were obtained

by

b I t

O0

using '4C- or 3H-aminoacid-labeled virus

prep-xx arations (Table2).

-,MW2Assuming that the gram molecular weight of

X-

O La Crosse viral RNA is 5.1 x

10",

it was

con-? 5_ cluded that the gram molecular

weight

of the

i

total virion proteins was 180 x

10'.

From the

2 percentageofvirionproteininL, Gl,

G2,

andN proteins, the amount of each protein present per virion wasdetermined

(Table

2). The

aver-age number of molecules of each protein per

virion was then calculated by dividing these

amountsby their respective molecularweights (Table 2).

C ,' It was apparent from these results that the

9

- numbers of Gl and G2 molecules per virion

wereapproximatelyequal (Table2).Itwasalso

4 concluded that the mass ofN protein

by

com-i

I,f, parison to the mass ofvirion RNA was ofthe

o

o0

orderof

10:1.

x 6 Absence of phosphoproteins in La Crosse

A virus

preparations.

A concentrated

prepara-a- : 0 tion of La Crosse virus, labeled by

[32PIphos-Il

1- 2 t FIG. 7. KT-GLY gradient centrifugation of bro-l bro-l melain-treated La Crosse virus. Virus labeled with

3_

iodine, aminoacids,orglucosamine was incubated

with (A) orwithout(A)bromelain(1 mg/ml)at35°C for 2 h. Each sample was rebanded in KT-GLY gradientsat40,000 rpm for8 h in an SW41 rotor. Fractions (0.4 ml) were collected from each gradient

A, andcounted. Densitiesweredeterminedbyweighing

0.10-mlsamplesat roomtemperature.(A) '25I-labeled 40 20 TOP La Crosse. (B)[14C]-or

P'Hglucosamine-labeled

La

FRACTION

NUMBER

Crosse.(C) 14C-or:H-aminoacid-labeled La Crosse.

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994 OBIJESKI ET AL.

2

2

x

I

2

-20 40 60 80 +

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FRACTION NUMBER

FIG. 8. Electropherogramsofproteins inthesupernatantand pellet fractions of La Crossevirus treated with(A)2%(vollvol) TritonX-100in 1MNaCI, (B) 2% (vollvol) TritonX-100in 0.5MNaCI,or(C) 2%(voll

vol) TritonX-100. Purified La Crosse virus labeledwith :H-amino acids and suspendedin 0.01 M Tris-hydrochloride buffer,pH 7.8,wasincubated withTritonX-100inthepresence orabsence of NaCl for20min

at20°C.Each preparationwasseparatedintosolubleand particulate fractions bycentrifugationat100,000 x

gfor2 h.Theproteins inthesupernatantorresuspended pelletfractionswereprecipitated withtrichloroacetic

acid (5% [vollvol] final concentration) after 100 jAg ofbovine serum albumin was added. The protein

precipitateswerewashedwithethanol, dissociated by SDS, andresolvedbycont-SDS-gelelectrophoresis. phate (1 x 107 cpm in 0.1 ml of TSE buffer),

was adjusted to 1% (wt/vol) SDS to solubilize the viral membrane and release the virion RNA. It was then diluted 50-fold with TSE

bufferand incubated with 0.5mgofpancreatic

RNase at 37°C for 1 h in order to digest the virion RNA. The viral proteins were

precip-itated at 4°C with 5 volumes of 5% (wt/vol) trichloroacetic acid. The precipitate was then

washed twice with5% acidto removeresidual

acid-soluble nucleotides and three times with ethanol to remove residual phospholipids and

acid. After dissociation by SDS, the

proteins

wereresolved by electrophoresisin an8% (wt/

vol) polyacrylamide gel, stained by Coomassie brilliantblue, destained, and then sliced into

1-mm sections. Although all four viral proteins were identified in the stained gels, no 32P was

IM NaCI

II -~ASupernatant

o-I-o Pellet

1A

I' II

NO5 NaCI

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LA CROSSE VIRUS 995

FRACTION NUMBEC FRACTIONn

12-0.

1 12

(.20.20 2 TPI0 40 6 8

FIG. 9. Isolation ofLaCrosse viral nucleocapsids. Apurified preparationof [3H]uridine (O)- and

14C-aminoacid WA-labeledLa Crossevirus was treatedfor20 min at20°C with2%(vollvol) TritonX-100in thepresence of0.01 MTris-hydrochloridebuffer,pH7.8,and1 MNaCl.Themixturewascentrifuged for

24hat4°C and40,000rpminacombinationKT-GLYgradientcontainingIMNaCl(A). The distribution oflabelineachof thirty0.4-mlfractionswasdeterminedaswellasthedensityofselectedsamples.Pooled

firactions

(IorII)ofthegradient weredialyzedfreefrom potassium tartrate; theproteinswererecoveredby

precipitationwith5%(wtlvol) trichloroaceticacid,and theprecipitateswere washed with acid and ethanol.

Theproteins weredissociatedbySDS and2-mercaptoethanolandresolvedbycont-SDS-gelelectrophoresis

(B,fractionI; C,fractionII).

recovered in any part of the gel (data not shown).

DISCUSSION

Four proteinshavebeen identified in

prepa-rations ofLa Crossevirus. Two of theproteins

areglycoproteins (GlandG2). Bromelain treat-ment and iodination studies have shown that these glycoproteins are present in the outer

surface of the virus particles. Stoichiometric

considerationsindicatethattheGlandG2 pro-teinsarepresentinequimolaramounts (Table

2). Ofthe two other virionproteins, oneis the

majorcomponentof the viral nucleocapsids (N protein); the other (a large protein, L), also associated with the nucleocapsid, is present in limitedquantities.Themolecular weightsof La Crosse virus proteins (Table 1)wereestimated on the basis ofcomparative electrophoresis in

which three different gel systems with VSV Indiana structural proteins (30) and seven

other nonviral proteins ofknown size (Fig. 3)

wereused.

In investigations of La Crosse virus, Mc-Lerranand Arlinghaus(9) identifiedthree

ma-jorstructural proteins (molecular weight = 85

X 103, 45 x 103, and 26 x 103). Two glycopro-teins (molecular weight = 84 x 10: and 31 x

103) and athird protein of about21 x 103

dal-tonswere identified inOriboca virus (20),

an-othermemberof thebunyavirusgroup. Rosato

and associates (19) showed that each ofseven

bunyaviruses (Bunyamwera, California

en-cephalitis BFS-283, Tahyna, Oriboca,BeAn 17,

BeAn 974, and Murutucu) contained three pro-teins ranging in size from 85,000 to20,000 dal-tons. Morerecently, White (32)identifiedfour

structural proteins in California encephalitis

virusBFS-283. Threeof the proteins (molecular weight = 30 x

103,

38 x 103,and 82 x 103) were

glycoproteins; the fourth (molecular weight =

17.5 x 103) was associated with the virion nu-VOL. 19X 1976

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TABLE 2. Protein compositioni of La Crosse virus

Total protein('1) Molwt Daltons of protein/ No. ofmolecules/

Proteinspecies - virion virion

Incorporation' Stained (x 10 (x 10)

L ... 5 3 180 4.6 25

GI ... 50 51 120 78 650

G2 ... 14 14 34 21.4 629

N ... 31 32 23 48.9 2,126

Molecular weight estimates are based on the cont-SDS results given inTable1.

Total daltons of virion protein were determined from the 30:1 ratio of virion protein to RNA and the estimatethat the genomecomplexity of La Crossevirus is 5.1 x 10` daltons.

" Numberofprotein molecules per virion was calculated for each protein species bydividingthedaltonsof

protein per virion by their respective molecular weights.

Percentage of total protein fromincorporation studies represents the average of six separate determina-tionsinvolving:H-aminoacid- or'4C-amino acid-labeled virus(minimum3Hpergel = 9 x 104 cpm; mini-mum '4C pergel = 2 x 10'cpm).

Percentage oftotalproteins from a stained gel represents the average ofthreeseparatedeterminations of thearea under each peak.

cleocapsid. Uukuniemi virus, atick-borne

bun-yavirus-like isolate, serologically distinct from the bunyavirus group of viruses, was initially reported (18) to possess an envelope protein (molecular weight = 70 x 101) and another protein (molecular weight = 25 x 10:"). In

fur-ther analyses (2), involving resolution of the virion polypeptides by 15'/k polyacrylamide gels, two envelope proteins were identified.

Our estimate of the size of the largest La Crosse virus, glycoprotein (GI) (Table 1), is some 40,000 daltons larger than the estimate othershavefound for this protein in La Crosse and other related viruses (see above). Its size was determined by comparing its electropho-reticmobility with that of

/13-galactosidase

(mo-lecular weight = 130 x 10:1)and phosphorylase A (molecular 'voight = 92.5 x 10:;) as well as

other viral and nonviral proteins. The La Crosse Gl protein migrated in three gel sys-tems slightly faster than /3-galactosidase but slower than phosphorylase A, and was there-fore estimatedto be about 120,000 daltons. We

(J. Gentsch, D. H. L. Bishop, and J. F.

Obi-jeski, unpublished data) have foundone minor

and three major proteins associated with

snowshoe hare, Bunyamwera, and Main Drain viruses, all members ofthe bunyavirus group. Compared withtheresults reported here, rela-tively minor (or no) differences were found in the apparent size ortypes ofproteinsobserved in these three viruses.

Minor quantities ofa very large protein (L) werealways observed in La Crosse virus (and three otherbunyaviruses) inall ofourpurified

virus preparations. Its significance is not known; however, it may represent a virion-associated cellular protein or an uncleaved viralpolypeptide. Alternatively, itcould repre-sent a nondissociable polymeric aggregate of the othervirion proteins(GI, G2, and N). This

isunlikely, however, sinceL is notdissociated to any appreciable extent by boiling purified virions in 2.5% SDS, 8 M urea, and a vast excess of mercaptoethanol (5%). Similarly, re-duction and acetylation of thevirusproteinsdo not causetheamountofL todiminish, suggest-ingthatthis protein is not asimple aggregate. Ithasnotescapedourattention, however, that forrhabdoviruses a large protein is believed to be avirion-associated transcriptase component (30). The function and derivation of the La Crosse Lprotein remains to be determined.

Theestimates ofthe number of protein mole-cules per virion are based on the assumption that the sum molecular weight of the viral RNAis5.1 x 10'. InourstudieswithLaCrosse, snowshoe hare, Bunyamwera, and Main Drain viruses, we have identified three virion RNA components ofapproximate molecular weights of 2.9 x 10, 1.8 x 10', and 0.4 x 10' (manu-script in preparation). Although these RNA speciesareregularly presentinallofourvirus preparations (except when defective interfering particles are present; unpublished observa-tions), we have no evidence to prove that all three species are necessary for La Crosse virus to be infective. Other investigators have also identified segmented RNA genomes for La Crosse virus (9) and Uukuniemi virus (17). If, however, all of the genetic information resides in the largest RNA molecule, our estimates of the numberof protein molecules per virionwill betoo high by almost a factor of2.

ACKNOWLEDGMENTS

We thank Marianne D.Stappand Richard A. Klimas for excellent technical assistance and John Hierholzer for de-termining thesedimentationvalue of La Crosse virus.

Thisstudy wassupportedinpartby Public Health Serv-ice grantAI-13402, from theNationalInstitute ofAllergy and InfectiousDiseases, toD.H.L.B.

996

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LA CROSSE VIRUS 997

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