Junin virus structural proteins.

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Junin

Virus

Structural Proteins

Z. M. DE MARTINEZ SEGOVIA* AND M. I. DE MITRI

Divisi6nBioquimica, Departamento de Virus, Instituto Nacional de Microbiologia 'Carlos G. Malbrdn," Velez

Sarsfteld

563,BuenosAires,

Argentina

Received for publication 30 August 1976

Polyacrylamide gel electrophoresis ofpurified Junin virus revealed six

dis-tinct structural polypeptides, two major and four minor ones. Four of these

polypeptides appearedtobe covalently linked with carbohydrate. The molecular

weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the

most prominent protein and was nonglycosylated. The other major protein

(number 5), with a molecular weight of 38,000, was a glucoprotein and a

componentof the viral envelope. The location on the virion of three additional

glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with

aprotein with a molecularweightof 25,000, was not welldefined.

There is a remarkable unifornity in the mor- coldamino acids reduced to one-eighth of the

regu-phologyand nucleic acid composition of differ- lar level. (Cells were incubated with radioactive

entarenaviruses: virusparticles are round and precursorsafter day3postinfection.) To label viral

pleomorphic and have ribosome-like granules carbohydrates, radioactive glucosamine (1 ,uCi/ml)

in varying amounts (9). Lymphocytic chorio- was added to the medium 48h

postinfection.

meningitis, Pichinde, and Junin viruses share Virus

purification.

Topurify releasedvirus, the

commeingbit

.

'

.

infected fluidscollected fromdays4, 5,and 6 postin-commonbiochemical properties: all contain sin- fectionwere pooled and centrifuged at 1,500 x g to

gle-strand RNA and consist of fourmajor dis- remove cells and cell debris. The virus was then

cernible componentsand three minorones (1, 2, pelleted by ultracentrifugationat 100,000 x g for 3

6). Withregard to the amount of virion polypep- h. A virus pellet was then suspended in 0.05 M

tide in these arenaviruses, onthe other hand, sodiumborate buffer(pH7.2)-0.25Msucrose(1mlof

there are some discrepancies: Pichinde has four bufferper500 ml oftheoriginal volume).Aftersonic

polypeptides (8), and six werereported for

lym-

treatment for 2minat 10 kc/sin a Raytheon

oscilla-phocytic choriomeningitis virus (7). tor, theresuspended material wasbanded in a

dou-The purpose of this report istodescribe the ble_M_{sodium borate buffer}cushionconsistingof 50_(pHand 20%_7.2)_and_centrifugedsucrose in0.05_for

results obtained by studying the Junin virus 3 h at 40,000 rpm ina Spinco

SW50

rotor. The

virus-polypeptides tofindouttheirnumber, molecu- containing band, located at the

interphase,

was

re-lar weight, and structural identity. covered from the gradient and rebanded

isopycni-MATERIALS AND METHODS

cally

in a

preformed

25to 65% linear

density

gra-dient ofsucrosefor 7 hunder similar conditions of Virus, cells,and medium. The procedurefor the centrifugation. Twenty 0.25-ml fractions were col-preparation ofmedia, cells, stock virus pool, and lected by bottompuncture of the tube. In each sam-virustitrationhas been described previously (5). ple, the infectious virus titer and theradioactivity Infection of cells andlabelingof virus. Twenty- weredetermined. These samples could be stored at four-hourmonolayers ofBHK-21 cells in Roux bot- -70°C for long periodswithout loss of infectivity. tles or inroller flasks (100ml) were infected with This procedure recovered30% of theoriginal virus stock virusat amultiplicityof infectionof approxi- infectivity.In some instances,infectivitypeak frac-mately 3 50% lethal doses (LD50) per ml percell. tionsfrom thefirstsucrosegradientwere pooled and After 3 h ofadsorptionat 37°C, the inoculumwas diluted with 0.05 M sodium borate buffer (pH 7.2), removed and the cells were washed with 0.01 M and a1.5-ml amount of this suspension was layered phosphate-buffered saline,pH 7.2, fed with 40 or 100 into a discontinuous gradient containing 0.5 ml of mlof maintenance medium, and incubated at 37°C. 40%, 1.3 ml of 30%, and 1.3 ml of 25%(wt/wt) CsCl in

Each 24-h monolayerwaswashed withphosphate- thesamebuffer. Thegradientwascentrifugedfor90

buffered saline, and new medium wasadded. The minat 40,000 rpm in a Spinco SW50 rotor.

infected fluids were collected daily from days 4 to 6 Polyacrylamide gel electrophoresis. The gradient postinfection.Forisotopiclabelingof viralprotein,1 fractions, chosen becauseof the coincidence of maxi-,uCi of 14C-or3H-labeled aminoacid mixture per ml mumradioactivity andinfectivity, werecentrifuged was added to the medium with a concentration of at40,000 rpmfor 3 h in aSpinco SW50rotor. The

579

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resultingpellet was dissolved in a solution contain- 16 ing 1%(wt/vol) sodium dodecyl sulfate (SDS) and 1% 15 (vol/vol)2-mercaptoethanol according tothe

proce-dure ofMaizel (4). After heating at 100°C for 1 min 14

-the sample was applied directly to -the gel in a vol-

13/

ume ofapproximately 200

,l,

with5 mgof bromo-

1ff

phenol

blue asthe

tracking

dye

and 10% sucrose. 212 l8

Electrophoresis was carried out in a 13-cmcylindri- x11

cal gel for 30 min at 40 V and for an additional 6 h at i7;

90 V.The running buffer was 0.1 M phosphate with 6o

0.1% SDS. After electrophoresis, the gels were re- X9 X -6!E

moved from the tube.Samplesthat did not contain N

radioactive protein werestainedfor 2 h in 1% amido " -50

Schwarz in 7%(vol/vol)aceticacid and destainedby a 7 l diffusionfor 48 hwith several changes of acetic acid. C

Gels containingradioactive protein were frozen and I 6 4

fractionated into 2-mm slices. The slicesweretrans-

S-|1

5 ferred to countingvials,and each one was dissolved K

with 0.5 ml of 100 volumes ofhydrogen peroxide '' V\

du-ingovernight incubation at 600C. Then 4.5 ml of 3 2

toluene-based scintillation fluid was added, and

2-samples were counted in a Tri-Carb scintillation 1

spectrometer. In all experiments 10% gels were 1

used. 0

Standardproteins fordetermination of molecu- 1 2 3 4 5 6 7 8 9 1O larweight. Thefollowingproteins, purchasedfrom

traction

number

MannResearchLaboratories, wereused as markers FIG. 1. Step gradient purification of "4C-amino

for molecular weight determination: catalase, acid-labeled Junin virus. Concentrated virus (1 ml) 94,000; bovinealbumin, 68,000; ovalbumin, 45,000; was layered on top of a double cushion consisting of trypsin, 23,800; andhemoglobin, 16,000. 50 and 20%sucrose in 0.01 M sodium borate buffer (pH 7.2). The virus was centrifuged in a Spinco RESULTS SW50rotor at 40,000 rpm at4°C for 3 h. Fractions

Virus purification. WhenBHK-21cells were were collected from the bottom of the tube and

ana-infectedata m ,of. ...infection of3LD, lyzed for infectivity (0) and acid-insoluble

radioac-pieectel

at

a mu

lip

licity

3I

on

OI J

..r

tivity (@).Purifiedfluidsfromlabeled

mock-infected

per cell, maximum virus titers of 10-8 LD50 per cells weremixedwith infected, unlabeled fluids and

0.02mlwereconsistentlyobtainedondays4, 5, centrifuged under identical conditions (x).

and 6 postinfection. Junin virus was

concen-trated and purified from the cell-free medium Electrophoresis pattern of proteins from

asdescribedabove.Figure1showsthepurifica- unfractionated Junin virus. Figure 2 (upper

tion of Junin virus labeled with a "4C-amino panel) shows a photograph and densitometer

acid mixture in a double-cushion gradient. profiles of Junin virus proteins subjected to

Fractions were analyzed for both infectivity polyacrylamide electrophoresis after the gel

and radioactivity. The infective viruswas dis- was stained with amido Schwarz. Purified

tributed in abroad zone of the gradient, indi- preparations of virus reproducibly yielded six

cating heterogeneity ofsize. Maximum infec- bands, which have been designated

numeri-tivitybandedat aslightlygreaterdensitythan callyfrom theorigin. Twobands,3and5, were

maximum radioactivity. Similar results were staineddensely. Thewidth of band5indicates

reported by Carteretal. (2) andwereobtained that thiscomponent isheterogeneousin

molec-inourlaboratory whenvirus waslabeled with ular size. Minor additional

low-molecular-[3H]uridine(1).This displacement could be due weightpolypeptides werefrequently present.

tothepresenceof defectiveinterferingparticles To obtainmore detailed infornation on the

at the peak of maximum radioactivity. When Junin virus polypeptides, an analysis was

labeled mock-infected BHK cells were mixed made withradioactively labeled viruses. There

with unlabeled infected fluids and processed was agood correspondencebetweenthepattern

under identicalconditions,noradioactivitywas ofpolypeptides instainedgels and thepattern

foundinanyof the gradientfractions. obtained withviruslabeled with"4C-aminoacid

Asecondstepofpurificationwasachievedby mixtures (Fig. 2, lower panel). Also, six

pro-isopycnic density gradient centrifugation. At teins werepresent, two in large amounts

(pro-thisstage, therecoveryofinfectivitywas near teins 3 and 5). Protein 3 was the most

promi-30%that of theoriginal fluids. Titers ofapprox- nent. A variable amount of polypeptides of

imately loll to 1012

LD,O/ml

were usually ob- lowermolecular weight was detected in

differ-tained. entpreparations.

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VOL. 21, 1977 581

12 3 4 5 6 SDS-containing polyacrylamide gel,

polypep-tides are separated largely on the basis of

mo-lecular size, the distance migrated being

lin-early related to thelog of the molecularweight.

The corresponding slope and the intercepts were obtained by the least-squares method, us-ing protein markers of known molecular

weight. By interpolating in the resulting

straight

line, the molecular

weight

of each

pol-ypeptide was deternined.

The weighted mean of the molecular weight

dw of each Junin viruspolypeptide from three

in-dependent experiments was found to be 91,000,

3 71,000, 64,000, 52,000, 38,000, and 25,000,

re-ffi spectively, with a percentage of error never

2 1 2 5 6

higher

than6%

(legend

of

Fig.

2).

1111J l

Glycoproteins

of Junin virus. Todetermine

which Junin virus proteins contained

carbohy-6 l drates, the virus was labeled with 3H-amino

o l acids, purified as described in Materials and

x l Methods, mixed with purified virus labeled

E 1 Z ^ with[14C]glucosamine,and subjected to

electro-10 Al j Iphoresis in polyacrylamide gel. The most

prom-inent protein (peak 3) was apparently free from

14C label

(Fig.

3). Four

polypeptides

were

iden-i I , . .

10 20 30 40 so 3

fraction number

FIG. 2. Analytical polyacrylamide gel electro- 20

-pherograms of dissociated proteins of Junin virus. 1 2 4 5 6

Purified virions were disrupted with SDS and 2- |

mercaptoethanol and subjected to electrophoresis for I I 1 1 6 hat 90 V as described in Materials and Methods.

Upperpanel shows a photograph ofa gel stained with

1 %amido Schwarzand, directly above, its densitom- X IS- x

etertracing. Lowerpanel shows an electropherogram E E

ofpolypeptides ofpurifiedJunin virus grown in BHK 1o cells incubated with '4C-labeled amino acids. After

electrophoresis, the gels were frozen andsliced,and , c

the radioactivity of each slice was determined. The 10- _ mE

position of each of the six viral polypeptides is indi- *

cated by arrows. The virus-specific proteins have E 0i

been assigned numbers from 1 to 6. In this and i subsequent gel patterns, the origin is on the left and l

the anode on the right.Proteinstandards, specified 220

inMaterials and Methods, were co-electrophoresed in

order to determine the molecular weight of the six 6 differentpolypeptides. The weighted mean ofthe

mo-lecularweight of each polypeptide from three inde- i

pendent experiments was found to be: protein 1,

91,450 4,208; protein 2,71,680 +3,418;protein 3, -0-0-0---64,233 ±3,129;protein 4,52,456±2,658;protein5, 1a020 30 40 50 38,315 +2,089;andprotein 6, 25,477 + 1,548.

fraction

number

FIG. 3. Analytical SDS-polyacrylamide gel

elec-Estimation of molecular weights. The mo- trophoresis of the structural proteins of Junin virus,

lecularweightsof theviralproteinsweredeter- labeled with

[f4C]glucosamine

and 3H-amino acids.

mined by the method ofShapiroet al. (10) b Theprocedurewasthesame asthatdescribedin the mined by the method of Shapiro et al. (10) by legend of Fig. 2. After electrophoresis, the gel was

comparing theirelectrophoreticmobilities with frozen and sliced, and the radioactivity of both

the migration ofaseries ofproteins of known ['4C]glucosamine (0) and 3H-labeled amino acids

molecular weights. By electrophoresis in an (O)wasdetermined.

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[image:3.501.47.235.56.356.2] [image:3.501.252.440.329.581.2]

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tified asglycoproteins (peaks 1, 2, 4, and 5). The large amount of protein was solubilized and

largest glycoprotein peak corresponded to pro- could be detected in the lighter region of the

tein 5. Occasionally this glycoprotein was dis- gradient along with a significant amount of

tributed in two adjacent bands. The heteroge- [3H]uridine. We can assume that in our

experi-neous material labeled with glucosamine that mental conditions the detergent treatment had

wasseen near theanode could represent glyco- altered the virion integrity, that someparticles

lipids. had been broken, but that nevertheless some

FractionationofJunin virus with Triton X- fairly large structures remained intact. In

100. To relate the polypeptides observed in these structures, one or more polypeptides

re-acrylamide gels to structural virus components, mainedassociated with virus RNA.

purified virus labeled with a 14C-amino acid Figure5 (upperpanel) shows the radioactive

mixture and [3H]uridine were disrupted with profile of these polypeptidesonpolyacrylamide

Triton X-100 to a finalconcentration of 2% in gelelectrophoresis, and this profile was

super-thepresenceofdithiothreitolandNaCl,accord- imposedon the pattern obtained with the

un-ing to the procedure of Emerson and Wagner treated virus. From this figure, we can

con-(3). After 30 min at 37°C, theinfectivity of the clude that protein 5 was quantitatively

ex-virus sample wascompletelyabolished. tracted from the virion. When a double label

This treated virusbehaveddifferentlyince- was used (3H-labeled amino acids and

sium chloride density gradient than did the

untreatedone. Mostof the

[3H]RNA

andsome 3

14C-labeled proteins remained associated in a I

particle which, in turn, was displaced toward

X0

r

X2

4 5 6 _50,

the denser zone ofthe gradient. A relatively o I| I

2

0.~~~~~~~~~~~~~~~

15 5

2250e

30

A

10 ..r

~50-

601

0.r

frjCT,On

B b0Electropherograms of0 0 the Junin0

viruspr

30LJ

FIG. 4. Sedimentation in a cesium chloride gra- teins isolated afterTriton treatment. Fractions 7 and dientoTrtntetdaduntreatedcontrol virions. 8, from a CsClgradient similar to theone'shown in Purified virus labeled simultaneously with Fig. 4B, andfractionl11 (Fig. 4A) wereprepared for [3H]uridine (O---O) and '4(2-labeled amino acids electrophoresis on 10% acrylamide gels as described

(-)wastreated with TritonX-IOOasdescribed in the in Materials and Methods. The profiles obtained

text. Virustreated with sodium borate buffer under from thecontrol(0---0)and Triton-treated prepa-the sameconditionsservedas a control. Thetreated rations (0 0) were superimposed and appear in

virions were rebanded in a discontinuous cesium the top panel. The profiles obtained with a virus

chloride gradient as indicated in Materials and labeled with['4C]glucosamine (-@*) and 3H-la-Methods. Fractions were collected, and the radioac- beled amino acid mixture (0 0) and processed

tivityofeach wasdetermined. (A) Control virus; (B) with Triton X-100 under identical conditions are

Triton-treatedvirus, shown in the lower panel.

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[image:4.501.270.453.169.534.2] [image:4.501.69.249.308.546.2]

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['4C]glucosamine), analysis of the treated virus of the virus and remain inseparable by any of

gave identical results. The major glycoprotein the methods described. If this is the case, it

was entirely solubilized, but the remaining should be possible to detect this by producing

three glycoproteins were still present (Fig. 5, radioactively labeled cell membrane fragments

lowerpanel). The most prominent nonglycosyl- from mock-infected BHK cells, mixing them

atedprotein remained in thedetergent-treated with unlabeled virus, andpurifying the virus.

viruswith the same electrophoretic mobility. We have seenthat no labeled virus was found

DISCUSSION inthe

first

step

of

purification

(Fig.

1),

indicat-ing that it is unlikely that cellular

contami-The results of the present study demonstrate nantscontributesignificantly to the viral

poly-that Junin virus is composed of at least six peptide pattern observed. Even if

contamina-polypeptides, twomajor and four minor ones. tionwere ruled out, we wonder how many of

The most prominent polypeptide, with a molec- theproteinsresolvedhereare virusgene

prod-ular weight of 64,000, remains in the detergent- ucts or cell gene products. It is very possible

treated virus with the same electrophoretic mo- thatribosomal proteins are responsible for the

bility. We can assume that this protein,cosedi- smallproteinsfound in theregion

oflow-molec-menting with Junin virusRNA, is the nucleo- ular-weightpolypeptides.

protein of the virion. The second prominent

polypeptide, withamolecularweight of34,000, ACKNOWLEDGMENTS

isanenvelope glycoprotein accessibletodeter- This workwassupportedinpart by grants from

Funda-gentextraction. Recent workinthislaboratory ci6nEmilio Ocampo, ConsejoNacional de Investigaciones

has suggested that this protein is involved in Cientificas_Ciencia_y_TecnicayTecnicas(grant60586-75), andSecretariade

(grant6655/74).

eliciting neutralizing antibodles (B.Crestaand We thank Liliana Gonzalez, NildaTasc6n, and Paula

Z. M. Segovia, unpublished data). The biologi- Padulaforexcellent technicalassistance.

cal activities and the location of the four

poly-peptides have not yet been identified. Since LITERATURE CITED

glycoproteins are verypossibly structural com- 1. Afton,M.C.,0.Grau,Z.Martinez Segovia, and M. T.

ponentsof theenvelope, itseemsclearthat,in France Fernandez. 1976. RNA composition of Junin

ourhands, Triton X-100disrupts, butdoesnot virus. J. Virol. 18:833-838.

2. Carter, M. F., N. Biswal, and W. E. Rawls. 1973.

entirelyremove, theenvelope. We have,onthe Characterization of nucleic acidof Pichinde virus.J.

other hand, observed that a nonglycosylated Virol. 11:61-68.

protein is often extracted by Triton X-100, an 3. Emerson, S. V.,and R. R. Wagner.1972. Dissociation

observation also made by Emerson and Wagner andreconstitution of thetranscriptase andtemplate

y

,.,. ... . ,activities of vesicular stomatitis B and T virions. J.

(3) working with vesicular stomatitisvirus. It Virol. 10:297-309.

remainstobeseenwhether thesepolypeptides 4. Maizel, J. V. 1969. Acrylamide gel electrophoresis of

arelocatedwithin orbelow the lipid envelope. proteins and nucleic acids,p. 334-362. In K. Habel

The availability ofa method that completely and N. P.Salzman (ed.),Fundamental techniques in

removesthemembrane

should...

allow usto

avirology.

Academic PressInc.,New York.

removes the membrane should allow us to ac- 5. Martinez Segovia, Z. M., M. I.DeMitri, and S.

Bender-quire clear-cut inforrnationonthis issue. sky. 1974. Nutritionalrequirements for Junin virus

The molecular weights of the six proteins production inBHK culturedcells. ActaPhysiol. Lat.

total 344,000. The single-strand Junin virus Am. 24:656-661.

RNA,ithmolcula weihtof3.74x 10, (1, 6.Pedersen,I. R. 1971.Lymphocyticchoriomeningitis vi-RNA, with amolecular weight of 3.74 x 106 (1), Pei-I-usRNAs. Nature(London)New Biol.234:112-114.

cancode for differentpolypeptides withatotal 7. Pedersen,I. R. 1973.LCMvirus:itspurificationand its

molecular weight of3.74 x 105.Thismeansthat chemical andphysical properties,p.13-23. InF.

Leh-thecombinedmolecularweightsof Junin virus man-Grube (ed.), Lymphocytic choriomeningitis

vi-proteins

wo.ld

require approximately 92% of rusand other arenaviruses.Springer-Verlag,Berlin. proteins WOUld require approximately 92% Of 8. Ramos, B. A., R. J. Courtney, and W. E.Rawls.1972.

the coding capacity of the virus genome. It is Structural proteins of Pichinde virus. J. Virol.

conceivable that theglycoprotein withamolec- 10:661-667.

ularweight of

91,000

could be an aggregationof 9. Rowe, J., J. Murphy, G. H. Bergold, I. Casals, I.

Hotchin,K. M.Johnson, F.Lehmann-Grube, C.A.

two ormorepolypeptides, and moreover, some Mims, E. Traub, and P. A. Webb. 1970.

Arenavi-polypeptidesthatfrequentlyappearinthepro- ruses:proposedname for anewly definedvirusgroup.

file could represent cellular membraneproteins J.Virol.5:651-652.

purifying togetherwith the virus. Itmightbe 10. Shapiro, A. L.,_Molecular_weightE._{estimation of}Vinuela, and J. V._polypeptideMaizel. 1967._chains_by

supposed thatcellular membrane constituents electrophoresis in SDS-polyacrylamide gels.

Bio-could share all of thephysiochemical properties chem.Biophys.Res.Commun. 28:815-820.

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