Proteins Specified by Herpes Simplex Virus VIII. Characterization and Composition of Multiple Capsid Forms of Subtypes 1 and 2

Copyright@ 1972 American Society for Microbiology

Proteins Specified

by Herpes Simplex

Virus

VIII.

Characterization

and

Composition

of Multiple

Capsid

Forms

of

Subtypes

1

and

2

WADE GIBSON AND BERNARD ROIZMAN

Department of Microbiology, The UniversityofChicago, Chicago,Illinois60637

Received for publication7August1972

Two classes of herpesviruscapsids, designated A and B, wereisolated from the

nuclei of humancells infected with herpes simplex virus (HSV). A andB capsids

share incommonfour structural proteins, i.e., no. 5, 19,23, and24. Bcapsids

con-tain 7.7 to 9.7 times moredeoxyribonucleic acid than A capsids; moreover, they

contain proteins no. 21 and 22a in addition. All of the proteins contained in the

capsid except no. 22aare present in the enveloped nucleocapsids (virions) in

ap-proximatelythesamemolar ratios.The capsid proteins ofHSV-1 cannotbe differ-entiatedfromtheirHSV-2counterpartswithrespecttoelectrophoretic mobility. A

thirdclass of capsids, designatedC capsids, wasisolated fromvirionscontainedin the cytoplasm of infected cells by the same procedure used to obtain A and B

capsids.The Ccapsidscontain allof the proteinspresentin A capsids plus proteins

1to3and 21.

A preceding paper (25) inthis series reported on the composition, electrophoretic mobility,

and molecular weight of proteins comprising the herpes simplex virus (HSV) virion.

Subse-quently (13) it was shown that a number of the

glycosylated proteins present in the virion were

also present in purified membranes of infected

cells. The conclusion that these glycoproteins

were constituents of the envelope of the virion wasreinforcedbythe observation that NP40, a

mild nonionic detergent, partially stripped them fromthesurfaceof thevirion (25). The question

still remained astowhich proteinsare the

struc-turalcomponentsof the viral capsid.

Pertinent to the studies of the composition of

viralnucleocapsids reportedinthispaper arethe

following observations and reports. (i) The

nucleocapsid assembles in the nucleus, and in

cellsinfected withmost herpesvirusesit acquires

anenvelopeasit budsthrough the innerlamella of the nuclear membrane (3, 22). (ii) Electron

microscopy studies (18-20, 23, 24) have shown that at least two types of capsids are present

within the nucleus of HSV-infected cells, and thattheyappeartodiffer, atleast withregardto

staining properties, from the capsid contained within virions accumulating in the perinuclear

space orcysternaeoftheendoplasmicreticulum. (iii) The threemost currentdescriptionsof

herpes-virus nucleocapsids (1, 14, 21) have been based

on structures obtained by widely differing

tech-niquesand,notsurprisingly,arenotinagreement

about the exact protein composition.

Further-more, two of thethreedescriptions (14, 21) are

based on radioisotopic profiles obtained by

sectioning the analytical acrylamide gels-a technique that does not afford ashigh a

resolu-tion as absorbance profiles from either stained

gelsorautoradiograms.

Weare presentingevidence here insupport of

thefollowing: (i)two classesofcapsids,differing

withrespect tobothdeoxyribonucleicacid(DNA)

content and protein composition, are present in

the nucleiof HSV-infectedcellsand (ii) the

pro-tein composition of capsids derivedby stripping

of theenvelope from virions differs from that of eithertypeofintranuclear

capsid.

MATERIALS AND METHODS

Virus and cells. ThepropertiesofHSVsubtypes 1

and 2used in these studies have been described

else-where (7, 15, 25). Enveloped virions were produced

in HEp-2 cells, and the intranuclear capsids were

produced in either HEp-2 or Vero (simian) cells.

Pertinentinformation aboutthegrowth,maintenance,

infection, andradioisotopic labelingofcells hasbeen

presented in detail by Spear and Roizman (25).

Deviations from these general techniques are

indi-cated in thetext.

Isotopes and reagents. The isotopes and reagents

were reconstituted 3H-protein hydrolysate (mixture

no. 3130-08, 1 mCi/ml) and thymidine-2-14C (no.

2523-96, 10 /ACi/mi) from Schwarz/Mann,

Orange-burg, N.Y.; uniformly labeled 14C-L-amino acid

mixture (mixture no. NEC-445, 100 ,Ci/ml), and

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D-glucosamine-1-14C (NEC-193X, 100,Ci/ml) from New EnglandNuclear Corp., Boston, Mass.;

acryl-amide from K & K Laboratories, Plainview, N.Y.;

N,N'-methylenebisacrylamide and N,N,N' ,N'-tetra-methylethylenediamine (TEMED) from Eastman Chemicals, Rochester, N.Y.; dextran 10 from

Phar-macia, Uppsala, Sweden; Coomassie Brilliant Blue

fromColabLaboratories, Inc., Glenwood, Ill.;

ultra-pure urea from Mann Research Laboratories, New

York, N.Y.;NonidetP-40, agiftfrom ShellOilCo.,

New York, N.Y.; sodium desoxycholate from Schwarz/Mann, New York, N.Y.; BRIJ 58 (poly-oxyethylene [20] cetyl ether) from Atlas Chemical Industries, Inc., Wilmington, Del.; herring sperm DNA, typeIV,fromSigmaChemicalCo.,St. Louis,

Mo.; bovine serum albumin (crystallized A-grade)

from Calbiochem, Los Angeles, Calif.; and deoxy-ribonuclease I (electrophoretically purified) from Worthington Biochemical Corp., Freehold,N.J.

Isolation of intranuclear virus particles. Cells were

harvested between 18 and 24 hr after infection;

col-lected by centrifugation at 1,500 X g for 10 min (low-speed centrifugation); and resuspended (5 X 107 cells/ml) in 0.15 M NaCl, 0.01 M tris(hydroxy-methyl)aminomethane (Tris), pH 7.2, and 0.002 M MgCl2containing

1%c

NP40.After incubationfor 30

min at 0 C, the nuclei were sedimented from the

suspension bylow-speedcentrifugation andlysedby

adding sodium deoxycholate (DOC) to a final

con-centrationof 0.5%. Thelysatewasgentlywarmed in

the presence of deoxyribonuclease (50 ,ug/ml) to

decrease the viscosity. BRIJ-58 and urea were then

added to final concentrations of 0.5% and 0.5 M,

respectively, and the extract was clarified by

low-speed centrifugation. The supernatant fluid was

layered on top of linear, 10 to 40% (w/w) sucrose density gradients,prepared in 0.15 M NaCl and 0.01 M sodium phosphate buffer (pH 7.2), and centrifuged

for 60 min at 23,000 rev/min and 4 C in a Spinco

SW27 rotor. Two light-scatteringbandslocated near

themiddleof the tube wereresolvedduring the course ofcentrifugation. Each was recovered mechanically

fromthetopof the gradient and eitheranalyzed

im-mediately,orfrozen and stored at -20 C.

Isolation of enveloped virions. Enveloped virions

were isolated essentially as described by Spear and

Roizman (25). Briefly, infected cells were swollen in

hypotonic buffer and disrupted by several strokes

with a Dounce homogenizer, and the cytoplasmic

fraction was layered onto 3 to 30% dextran-10

gradients and centrifuged at 23,000 rev/min for 60

min in a Spinco SW25.3 rotor at 4 C. Enveloped

virions sedimentedto apositionjust above the middle

of the tube, and formed alight-scattering zone that

wascollected mechanicallyfrom the top of the tube.

Thismaterialwas not furtherpurified.

Preparation ofdetergent-treated cytoplasmicvirions.

(i) NP-40-extracted cytoplasmic virions were

pre-pared by suspendingapproximately 108infected cells

in 1 ml of 0.15 M NaCl, 0.01 M Tris (pH 7.2), and

0.002 M MgCl containing 1% NP-40. After

incuba-tion for 20min at 0 C the nuclei were removed from

the lysate by low-speed centrifugation. The lysate wasfurtherclarified by a secondlow-speed centrifuga-tion.

(ii) NP-40/DOC-extractedcytoplasmicvirionswere

prepared by further processing a clarified NP-40 lysate,obtained as above, with DOC (0.5%),

deoxy-ribonuclease (50 Mug/ml), BRIJ-58 (0.5%), and urea

(0.5M) in thesamefashionasdescribed above for the

isolation of intranuclear virus particles. The lysate

wasclarifiedby low-speedcentrifugation.

Both NP40 and NP-40/DOC lysates were

sub-jected to rate-velocity sedimentation on 10 to 40%

(w/w), linear sucrose density gradients prepared in

0.15 M NaCl and 0.01 M sodiumphosphate (pH7.2)

andcentrifugedat 23,000rev/min for 60 min at4 C inaSpincoSW27or SW25.3rotor.One visible band

wasresolvedfrom eachlysate atabout themid-point

of the tube; itwas collected mechanically from the

top ofthegradientand stored at -20C.

Analytical gel procedures. The electrophoretic,

staining, and autoradiographic techniques used in

these studies have beendescribed in detail bySpear

and Roizman (25). The important features are as

follows. (i) Samples to be analyzed were solubilized

by heatingat 100C for 2 minin the presenceof2%

sodium dodecyl sulfate (SDS), 5%

beta-mercapto-ethanol, and 50 mm Tris (pH7.0). Solubilized

prep-arations wereapplied to thegelsin 50- to 150-Militer

volumes generally containing between 50 and 100,ug

ofprotein. (ii) Discontinuous SDS gels (4, 16) were

formed in 12-cm glass tubes cleaned by soaking

overnight inasolution of concentrated sulfuric

acid-dichromic acid and rinsed thoroughly with water,

rather thansiliconizedasrecommendedin theoriginal

procedure. The main gel (8.5 cm in length) contained

8.5% acrylamide; the stacker gel (2.0 cm inlength)

contained 3% acrylamide. (iii) Proteins in the gel

were fixed and stained with 0.03% Coomassie

Bril-liant Blue in 10% acetic acid and 25% 2-propanol

according tothe procedureofFairbankset al. (10). Destainingwas in 10% acetic acid, 10% 2-propanol. (iv) Autoradiograms were made from gels sliced longitudinallyanddried asdescribed byFairbanks(9).

A mechanical press was used to hold the dried gel

firmly against the X-ray film during exposure. (v)

Absorbance profiles were made from stainedgelsand

autoradiograms, both scanned at 550 nm, with the Gilford linear transport attachment and Gilford recordingspectrophotometer.

Protein and DNA analyses. Protein concentrations

were determined by the Lowry procedure (17) as

modified by Eggstein and Kreutz (6). Bovine serum

albuminwasused as the standard.

DNA concentrations were determined by the

di-phenylamine technique described by Dische (5) as modified by Burton (2). Herring sperm DNA was

used as thestandard.

Electron microscopy. Thin sections were prepared

forelectronmicroscopyexamination from pellets of

A and Bcapsids.Briefly, capsidsuspensionsrecovered

from nuclear lysates as described above were

sub-jected to centrifugation at 25,000 rev/min for 2 hrat

4 Cin aSpinco SW27 rotor. Thepelletsthat were

ob-tained were fixed in gluteraldehyde, postfixed in

osmium tetroxide, embedded in Epon, and stained

withuranylacetateandleadcitrateprior to

examina-tion in anAEI-EM6Bmicroscope. A detailed

descrip-tion of this procedure and of the technique used to

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prepare wholemountsnegatively stained with sodium phosphotungstic acid has been published elsewhere (25).

RESULTS

Centrifugation of nuclear lysates. Nuclear ex-tractspreparedasdescribed above, on centrifuga-tionthroughadensity gradient, formedtwobands designated astop and bottom (Fig. 1). A third

band occasionally appeared near the bottom of

the tube; its occurrence generally indicated in-complete solubilization of cytoplasmic

mem-branes with NP-40. The bands shown in Fig. 1 were obtained from approximately 109 cells

infected withHSV-2.

Somevariability has been noted in theamount

ofmaterial in the top and bottom bandsrecovered from different

preparations;

however, several

lines of evidence indicate that it does not result

from the

extraction

procedureperse.Specifically, (i) extending NP-40 extraction to 24 hr at 0C or substituting Triton X-100 for NP-40,

(ii)

changing theNP-40 extractionto15minat37C, and (iii)

allowing

thefinalextracttostandfor24 hr at 0 C

prior

to centrifugation do not visibly affect the

yield

ofthe material in either the topor

bottom bands.

Electron microscopy. Electron

microscopy

studies revealed the presence ofcapsids in both

top and bottom bands. Enveloped or

partially

enveloped

particles

were absent. In general the

capsids

in each band were uniform in size and

appearance. In

negatively

stained

(sodium

phos-photungstic acid) preparations,

the

capsids

in the

top band werefilled with

stain,

whereas thosein the bottom band were filled only

partially,

delineating an internal structure

(Fig. 2A, C).

Examination of thin sections oftop-and bottom-band material

(Fig.

2B,

D)

stained with

uranyl

acetate and lead citrate showed that

top-band

capsidsappeartobe empty, whereasbottom-band

capsids

containa

centrally located, densely

stain-ing structure. Cross-contamination ofthe bands

was estimated by

particle

counts to be lessthan 10%.

Forthe sakeof

brevity

and

uniformity,

weshall designate the particles in the top and bottom

bands asA andB

capsids,

respectively,

without

differentiating between the terms

capsid

and

nucleocapsid.

DNA-protein

composition

of top- and bottom-band capsids. Two series of

experiments

were

done. In the first

experiment

DNA and

protein

composition were determined on an unlabeled preparation of A and B

capsids

obtained from cellsinfected with HSV-2.

Following

their isola-tionasdescribedabove,the

capsids

were

pelleted

byadditional

centrifugation.

Sequential

DNAand proteindeterminationsweredoneon each

pellet.

FIG. 1. Top and bottom bands in asucrose density

gradienit

obtained by centrifugation of a

niuclear

lysate

from HEp-2 cells

inzfected

withHSV-2. The lysatewas

layeredon top of a 10 to 40% (w/w) sucrose density

gradient prepared in 0.15M NaCIand0.01 .fsodium

phosphate (pH 7.2) and centrifuged at 23,000

rev/min

at

4Cfor60miii ina

Spinico

S W27rotor.

In the second experiment, approximately 4 X

108

cells were infected with

hSV-1

and labeled

with 3H-amino acids

(1

,Ci/ml) and

'4C-thymi-dine (0.4

,Ci/ml)

from 1 to 24 hr

pcstinfection.

The nuclear lysate prepared from these cells was

centrifuged on sucrose density gradients. After

centrifugation, the top and bottom bands were

collected; an equal volume (50

Mliters)

of each was precipitated with cold,

7%sY-

trichloroacetic

acid, collected by vacuum filtration on 0.45-,m HA nitrocellulose discs, dried, and counted in a

PackardTri-Carb spectrometer.

Theresultssummarized in Table 1 indicate that Acapsids contain7.7to 9.7times less DNA than B capsids. Analyses of the size, structure, and

density of the B capsid DNA by rate velocity

sedimentation in neutral and

alkaline

sucrose density gradients, and by isopycnic

centrifuga-tioninCsCl solutions, respectively, indicate that it has the same characteristics as that isolated

from mature virions reported previously from

ourlaboratory (11, 12, 15).

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HSV-SPECIFIED PROTEINS

I

/1

.is

FIG. 2. Electrontmicrographsof top (A andB)and bottom(CandD) band,HS V-2capsids. Wholemountswere

niegativelystained with2%7osodiumphosphotungsticacid containing 0.01% bovineserumalbumin(Aan2dC). Tkin-sectiontedpreparationiswerestained withuranylacetateandleadcitrate(BandD). Thecapsidsareapproximately

100 imn indiameter.

TABLE1. DNA and protein content ofA andBcapsids

DNA/protein of

Material analyzed DNA Protein DNA/protein DNA/protein of

Acapsids

Expt 1 (HSV-2)

Acapsids 9.27,ug 671

,/g

0.014 9.7

Bcapsids 40.43JAg 297 ,Ag 0.136

Expt2 (HSV-1)

Acapsids 58 counts'mina 1,194counts,/minb 0.048 7.7

Bcapsids 282counts/min 766counts/min 0.368

a

14C-thymidine

was used to label HSV-DNA.

bH-amino acid mixture was used tolabel HSV proteins.

V.,

Structural proteins of A and B capsids. The protein components of theAand Bcapsidswere identified on the basis of three szries of

experi-ments. The first series was a direct comparison

ofAand Bcapsid proteins and an estimation of theirmolarproportionality. For the comparison, 3 x

109

cells were infected with HSV-2 and labeled with '4C-amino acids from 4 to 20 hr

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postinfection. The nuclei were then isolated and

the extract was centrifuged in sucrose density gradients. The top and bottom bands were col-lected, and the capsids were solubilized and

sub-jectedtoelectrophoresis in8.5% polyacrylamide gels. Absorbance profiles of the stained gels and

of their respective autoradiographic images are shown in Fig. 3. Estimations of the molar

pro-portionality of the capsid proteinswere based on

staining analyses of three different preparations

of B capsids, solubilized and separated

electro-phoretically in 8.5 % polyacrylamide gels as

described above.Asshownin Table 2,the molar

proportions of B capsid proteins 5, 19, 21, 22a, 23, and 24 are approximately 12:4:1:16:8:1,

respectively.

Thepurpose of the secondseries was to

estab-lish the identityof the A and B capsid proteins.

This was accomplished by subjecting HSV-1, Bcapsid proteinsto electrophoresis alone and in an artificial mixture with partially purified,

en-veloped HSV-1 virion proteins. Absorbance profiles of the stainedgelsareshown in Fig. 4.

Theobjectiveof the thirdseries was to compare the proteins of HSV-1 and HSV-2 intranuclear

capsids. Electropherograms of the HSV-1 and

HSV-2,Bcapsid proteinsare shown in Fig. 5.

The results of these analyses can be

sum-marized as follows. (i) B capsids contain four

major proteins and several mino detectable by autoradiography (F

band). On the basis of

co-electrn

virionproteins (Fig. 4), we have i

of the

major

proteins asbeingno. The fourth major protein is not

virion and has been designated a

a

.0

.,0

TopBand

*Corossi B;l11irntBlue Stain*

X 30 50 70 90 10 3

Distance(mm)

FIG. 3. Electropherograms ofthe p)

in the top (A capsids) andbottom (E

isolatedfrom HEp-2 cells infected u

labeled beginning 4 hr postinfkction

acids. Absorbance profiles of both

BrilliantBlue-stainedgelsandoftheir.

[image:5.493.263.456.82.195.2]

imnagesareshow,z.

TABLE 2. Proteincompositionzof HSV-2 B Capsids

Protein Percent total Molecular Molar

no. protein in weightb ratio'4-SD

capsidaai SD (Xi10-3)

5 62.5 i 2.8 155 12.59 i 0.56

19 6.8 i 1.1 53 4.00 i 0.66

21 1.5 i 0.6 44 1.06 i 0.44

22a 20.5 i 2.8 38.8 16.50 4: 2.25

23 8.1 4 0.5 33 7.66 4 0.47

24 0.8 i 0.1 25 1.00 i 0.12

aAverage + standard deviation of four

de-terminations: two were made from gels stained with Coomassie Brilliant Blue stain and scanned

at 550nm, asdescribed in Materials and Methods;

the third was made from a gel fixed overnight in

25% isopropanol-10% acetic acid, stained with

Fast Greenstain (1% in water, pH 3.0) for 12hr,

destained overnight, and scanned at 620 nm; and

a fourth was made from one of the Coomassie

Brilliant Blue-stained gels scanned at 500 nm.

IDetermined as described in reference 25 on the

basis of migration in 6, 7, 8.5, and9%

polyacryl-amidegels byusing proteins of known molecular

weight as standards.

cCalculated by dividing the percent of the total

protein by the molecular weight and normalizing

with respect to the protein present in lowest

amount, no. 24.

ir components Based on its

electrophoretic mobility

in the

zig.

3, bottom "mixture"

gel

(Fig. 4),

we estimate that

protein

ophoresis

with 22a hasamolecular

weight

of

38,800.

dentified three Onlytwo of the minor Bcapsid proteinsoccur

5, 19, and 23.

reproducibly

in the samerelative amounts from present in the preparation to preparation; one of these cor-ts protein 22a.

responds

to virion protein 24 (Fig. 4). The

second has been tentatively identified as protein

21 because it iselectrophoretically indistinguish-Botom Band able from protein 21 and the amounts of protein in band 24 and putative band 21 are about the same, as shown both by staining (Fig. 4and 5) and autoradiography (Fig. 3). A similar

pro-portionalitywas found between viralproteins 21 and 24 inhighly purified preparations of virions

(reference 25, Fig. 6,

"4C-amino

acid

autoradio-gram).

(ii)

A

capsids

lack

protein

22a and the minor component tentatively identified above as pro-tein 21, but are otherwise indistinguishablefrom Bcapsidsin

protein composition.

(iii)

Three

principal

lines of evidence indicate

that the

proteins

listed above are structural

roteins contained

components

of the A and B

capsids

andarenot

I capsids) banids

impurities.

Briefly,

proteins

5, 19, 23,

and 24 are

vith HSV-2 and presentin

approximately

thesamemolarratios in

with '4C-amino A and B

capsids

and in virions

(Fig.

3 and

4)

the Coomassie asthose shown in Table 2 for B capsids. Secondly,

autoradiographic in one

experiment

the bottom-band material was layered on top of a discontinuous

density

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HSV-SPECIFIED PROTEINS

CoomassieBrilliantBlue stain

10 30 50 70 90

[image:6.493.42.227.72.391.2]

Distance (mm)

FIG. 4. Electropherograms of B capsid and virion

proteinsalonean1dinanartificial mixture. Three

com-panion gelswere subjectedtoelectrophoresis, onze

coiz-taimzingpartially purified virions (HSV-J virionzs),

an-other containingHSV-1 Bcapsidproteins, anda third

containingapproximately equalamounts of virionanld

B-capsidproteins(mixture). Absorbance profiles ofthe

CoomassieBrilliantBlue-stainedgelsareshown.

gradient, formed by layering equal volumes of CsClsolutions

prepared

in0.005 M sodium phos-phate (pH 7.1) with densities of 1.25, 1.35, and

1.9 g/cm3, respectively. The gradient was then

centrifuged for 14 hrat30,000 rev/min and20 C in aSpincoSW41 rotor. Adiscretebandformed below the initial 1.25 to 1.35 g/cm3 interphase and contained all ofthe proteins in the relative

amountspresent in the sucrosedensity gradient. Thirdly, the electrophoretic mobility ofthe pro-teinsisindependent of the host, HEp-2 or Vero

cells,in whichthey are produced.

(iv) The electrophoretic mobilities of HSV-1 andHSV-2, Bcapsidproteins are thesame (Fig.

5).

Analysis of the capsids produced by stripping virons of theirenvelope. These experiments were

designed tocompare the protein composition of

capsids produced by stripping virions of their

envelope with that ofA and B

capsids

isolated

from nuclear lysates.

Approximately 4 x 108 HEp-2 cells were

in-fected with HSV-1 and labeled beginning 2 hr

after infectionwith

'4C-glucosamine.

Three types

ofparticleswereisolated fromthecells 24 hrafter infection, as described above: partially purified

virions,

NP-40-extracted

cytoplasmic

virions,

and

NP-40/DOC-extracted cytoplasmic

virions.

Briefly, the partially purified virions were

iso-lated fromhalf ofthe infected cells. The remain-ing cells were treated with 1 %c NP-40 in 0.15 M

NaCl, 0.01 M Tris

(pH

7.2), and 0.002 M

MgCI2

for20minat 0 C. Thenuclei whichwere notlysed

by thisprocedure werepelleted bycentrifugation

at 1,500 x g for 10 min. Half of the clarified

NP-40lysate was notfurtherprocessed; the other halfwas treated

sequentially

with DOC

(0.5%c),

deoxyribonuclease (50

ug/ml),

BRIJ-58

(0.5%),

and urea (0.5 M) in exactly the same fashion as

nucleiwerelysed for the preparation ofA and B

capsids. The NP-40 and the NP-40/DOC-treated lysates were then banded by centrifuga-tion on sucrose density gradients as described

Coomassie Brilliant Blue Stain

9

223HSV-1

_Oj

MIXTURE

10 30 50 70 90

Distance (mm)

FIG. 5. Electropherograms ofHS

V-J

andHS V-2 B

capsidproteins. Companiongels

con2taining,

respectively,

HSV-J,

B capsidproteins (HS

V-i);

HSV-2, B capsid

proteins (HS V-2); and a mixture

of

approximately

equalamountsof

HSV-i

and HSV-2, Bcapsid proteins

(mixture) weresubjectedtoelectrophoresis.Shownhere

areabsorbaiiceprofiles oftheCoomassie Brilliant Blue-stainedgels.

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1-3

Coomassie BrilliantBlue

stain

19 23

C-Capsids

II ~~~~~~~~22a

B-Capsids

10 30 50 70 90

[image:7.493.62.253.67.321.2]

Distance (mm)

FIG. 6. Electropherograms ofB andCcapsids

iso-latedfrom HSV-J-infected HEp-2 cells. Absorbance

profiles oftheCoomassieBrilliant Blue-stainedgelsare

shown.

above. One band formedineachgradient andwas

collected and storedat -20C.

Analyses of these three types of particles

re-vealed the following. (i) NP-40 treatment alone reduced the specific activity (counts per minute

of"C-glucosamine permicrogram of protein) of the NP-40particleby 90% ascompared with the partially purified virions. The electrophoretic profile of the particles obtained after treatment ofthecytoplasmwith NP-40alonewasessentially thesame as that ofparticles produced by NP-40

treatment of highly purified virions previously

described (25). (ii) NP-40treatment followed by DOC extraction further reduced the specific activitytolessthan 2% thatofthevirion. Fig.6 shows the electrophoretic profile of the proteins

presentintheseparticles, designatedasCcapsids.

The data are noteworthy from two points of view. First, the C capsids contain all of the B capsid proteins except protein 22a. Secondly, they contain several proteins presentinthevirion (proteins1to 3) absentfrom the A and Bcapsids

and purified membranes. They lack, however, thenonglycosylated proteins 4 and22,aswellas

allof theglycoproteinspresent inpurifiedvirions and in the purified membranes. We should add that the electropherograms of C capsid proteins exhibited a number ofless well-defined and less consistently reproduciblebands betweenproteins

5and 19. Parenthetically, increasing the detergent concentrations from 0.5% to 1 % had little noticeable effect on the protein composition of the C capsid.

DISCUSSION

Inthis paper we are reporting the isolation and composition of three classes of HSV capsids. The salient features of the data, particularly as they relatetothe structure of the virion and its assembly, may be summarized asfollows.

(i) Nucleocapsids class A and B are derived from the nucleus of the infected cells. As discussed below, they had not been enveloped at the time of

isolation. The A and B capsids differ in four respects. First, they differ in hydrodynamic properties in that A capsids sediment more slowly than Bcapsids. Second, they differ

morphologic-allyinthat Acapsids lack thecoreassociated with

viral DNA (reference 8, Fig. 2B, D). Third, as would be predicted from the electron micro-graphs, they differ in DNA content. B capsid

contains 10 times more DNA than A capsids;

this DNA is hydrodynamically and structurally

thesame asthatisolatedfrom virions and previ-ously described (11, 12, 15). Lastly, A and B

capsids differ in protein composition. Although

A andBcapsids ofHSV-1 cannot bedifferentiated from their HSV-2 counterparts, with respect to the proteins present or their electrophoretic

mobilities,the Bcapsids ofboth subtypes have a

major protein (no. 22a) not present in the A

capsids. It is pertinent that protein 22a is not a

contaminant of the B capsid but rather a

tena-ciously bound, structural component of that

particle. This conclusion is basedon twofindings. First, itisassociated inthe sucrosedensity

gradi-entsonlywiththe bottom band; it isabsent both from the gradient fractions above and below the

bottom band. Moreover, it sedimentsthrough a 2Murea-20% sucrose cushion and bands in CsCl

solutions inassociation with the B capsid.Second,

protein 22a is synthesized and becomes a struc-tural component of B capsids in both HEp-2 and Vero cellslong afterhostsynthesisisshutoff.

(ii) The C capsids have been derived by extrac-tion of virions with the same reagents as those used to lyse the nuclei ofinfected cells. As evi-denced bya98% decrease in thespecificactivity

of

"IC-glucosamine,

this procedure removes

essentiallyallof theglycoproteins associatedwith the virions. The C capsids differ substantially

fromboth A and B capsidswith respect to pro-tein composition. This, basically, is the datum which led us to conclude that A and B

capsids

do not represent degradation products of

en-veloped cytoplasmic virions.The most

interesting

featureofthe Ccapsid is its retention ofspecific nonglycosylatedproteins throughoutthe

rigorous

5

VIROL.

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detergent-isolation procedure. Thus, whereas proteins 4 and 22 were completely removed, protein band 1 to 3 was nearly quantitatively

retained, None of these are found as structural

components of A or B capsids. Preliminary

studies based on pulse-chase experiments and some propertiesof the proteins suggest that

pro-tein 22a isa precursor toprotein 22. The nature

and function of proteins 1 to 3 are not certain.

As previously reported (N. Frenkel and B.

Roizman,Proc. Nat. Acad. Sci. U.S.A., inpress;

Proc.25th Annu.Symp.Fund. CancerRes. M. D. Anderson Hosp. Tumor Inst., in press), these proteins are synthesizedlaterthan theremaining proteins ofthevirion andcorrespond, in time of synthesis andamountofDNAtemplate required

for their synthesis, to species ofribonucleic acid

transcribed from thesame amount of DNA late

ininfection.

One hypothesis, which reconciles the data re-ported in thispaper, thetranscriptional program

of the virus (N. Frenkel and B. Roizman, Proc.

Nat. Acad. Sci. U.S.A., in press; Proc. 25th

Annu.Symp.Fund. CancerRes. M. D. Anderson

Hosp. TumorInst., in press),the time of

synthe-sis of viral proteins (Gibson and Roizman,

manuscript in preparation), and the observation

that in HSV-infected cells empty capsidsdo not,

as a rule, become enveloped (22), is as follows. Protein 22a is found in association only with capsids containing DNA; B capsids, containing thisprotein,arethen able to bindproteins 1 to 3

and subsequently become enveloped. In the processprotein 22a ismodified, perhaps cleaved,

such that it is subsequently removable fromthe

enveloped particles by detergent treatment. Ac-cording to this scheme, protein 22a is on or ex-posed to the surface of the capsid and, as previ-ouslysuggested (N.Frenkel andB. Roizman,see

in press references cited above), proteins 1 to 3

regulateand enableenvelopment.

(iii) We do not know at present whether A capsids are by-products or the precursors to B capsids. Themajor differences between A and B capsidsarethepresenceinBcapsids ofa"core,"

more DNA, and two additional proteins, no.

21 and 22a. If protein 22a, present in higher

abundance than protein 21,is indeed onthe

sur-face ofthe capsid, as we suspected based on its

disappearance from virions, it wouldfollow that the only protein which conceivably might be

associated with the DNA core of the capsid is

protein 21.

(iv) Thedatapresented inthispaperpermit us

to order the virion proteins reported in

preced-ing papers of this series into four groups as shown inTable 3. They are as follows: group 1

r.

.c

r.

._

- +

± +

C14~~~~~~~~~~

C1 + +

n-~++++ev

+ ++

+

+ -H+

-+ 4i

+~-n + +

<14-+ -H++

+ +-+

+

x + +

t + '.+

1 +

-I

-< -+

Qo o

ua._

i o Q

_E _

-0

-Y . *t

$L

Q

ECA e

Cono

u E ~'o

00v~~

U05,.<

a, o'

X

NO,X

Y 0sr

-ej - 0% -) e

VC.) C

°°C X~

-_ t_3

D,, OU :)

_s .o

gO

Wnn =:/: -N

~

1051

H +

! lll

u

In

2

w

3-1 Cd

14

.z

0

.-d

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http://jvi.asm.org/

comprises proteins associated with membranes, i.e., proteins 7, 8, and 11 through 18 (13, 25;

T. Savage, B. Roizman, and J. W. Neine, J.

Gen. Virol., inpress). We areledto suspect that bandno. 19 containstwoproteins of whichoneis glycosylated and associated with membranes whereas the other isnot glycosylated and associ-ated with capsids. As indicassoci-ated in the table, membranes isolated from infected cells contain

traceamountsof proteins 22, 23, and 24. Itisnot

presentlyclearwhether thesearespecific structural

components or adventitious contaminants of

purified membrane preparations. Group 2 com-prises proteins present in A and B capsids, i.e., proteins5, 19, 21, 22a, 23, and 24. Proteinno. 5

accounts for more than 60%' (w/w) of the pro-teinconstituents of thecapsids (Table 2). Group 3comprises proteins 1 to4 and protein22which

we suspect bind to the capsids and intercalate

between the capsid and the envelope. Finally, group4comprises several minorbutreproducible

componentsof the virion whichwehavenotbeen

abletoplace.Theseareproteins 6, 9, 10,and 20.

ACKNOWLEDG MENTS

These studieswereaidedbyU.S. Public Health Service grant CA 08494 fromthe National CancerInstitute,grantNP15Gfrom theAmerican CancerSociety, and grantGB 27356X from the National Science Foundation. W. G. is a U.S. Public Health Service predoctoraltrainee(Al 00238 from the Nattional Institute of Allergy andInfectious Diseases).

We thank J. Heine,D.Furlong,and P.Wicdncrfor assistance with the electronmicroscopy.

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