Irreversible conversion of the physical state of herpes simplex virus preceding inactivation by thermal or antibody treatment.

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0022-538X/81/050737-12$02.00/0 Vol.

Irreversible Conversion of the

Physical State of Herpes

Simplex

Virus

Preceding Inactivation by Thermal or Antibody

Treatment

KAZUO YANAGIt

Department of Virology, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108, Japan

Received11September 1980/Accepted 4 February 1981

The buoyant densitycharacteristics of infectious particles of herpes

simplex

virustypes 1and2were studied bycentrifugationin sucrose and cesium chloride

density gradientswith a high resolution and satisfactory infectivity recovery. It wasshown thattwo populations of infectious virions differing in buoyant density

coexisted, the differencebeing slight but definite. The ratio of heavy (H) to light

(L) viralparticles varieddepending upon the solute used, the strain of virus, and

the

cell

origin. Circumstancesfavoring degradation of viral infectivity tended to

increase the H portion. Incubationat37°C largelyconverted L to H, and heating at

450C

convertedall virionstoH without infectivity. The L to H conversion was

irreversible, and no populations intermediate between L and H were clearly

observed. Inactivation by UV light irradiation didnotaffect the density

pattern.

ThatH was not anartefactdue to penetration of solutes, osmotic pressure, viral

aggregation, orloss of the envelopewas shown experimentally. A difference in

theoutershape of particlesbetween negativelystained L and H populations was

demonstratedby electron microscopy. Both cell-releasedand cell-bound herpes simplex virus particlesgaveessentially thesameresultwith respect tothe above

characteristics. The effect oflimitingdilutions ofantiserum wassimilarto thatof mildthermaltreatment, in thatdenservirionsincreased parallel to a decrease in

less dense virions. Sensitization with early immunoglobulin G, composed mainly of

complement-requiring

neutralizing antibody, caused the density transition, and subsequent addition of complement resulted inafurther increase in the buoyant

density

of the sensitized virions. The DNA in virus

particles

neutralized with immunoglobulin G plus complement remained resistant to DNase treatment.

Possible

implications

of thephenomenaarediscussed.

The primary purpose of this study was to

determine whetheror notsome

physicochemical

changes in

herpes

simplex

virus

(HSV)

were

correlated withits inactivation. Asabase line forthis, the buoyant

density

of intactHSVwas

determined first. As tothe

buoyant density

of HSV

particles,

the values

previously

obtained varied

widely,

i.e., from 1.253 to 1.281 g/ml in CsCl (5), from1.194to 1.226

g/ml

in

potassium

tartrate,and from 1.233to 1.267

g/ml

in

potas-sium citrate solution

(13).

These differences in

buoyantdensitywerethoughttobe due

partly

to

different

cell

origins (13, 19)

and

partly

differ-entvirus strains

(5).

Insomeof theabove-cited

cases the virus

band

formed a

peak

broad

enough to suggest a

heterogeneity

of

particle

density (13, 21). This ledus to the

supposition

that a density change

might

take

place

along

t Present address: Institute of Basic Medical Sciences, The UniversityofTsukuba,Niihari-gun,Ibaraki-ken305,Japan.

with degradation of viral

infectivity,

because rapid inactivation of HSV inCsClwasfound

by

earlier workers(18,

21).

The

instability

ofHSV

wasovercome,

however, by

Lawrence's

modifi-cation,

inwhich

equine herpesvirus

wasfloated

upfrom the bottomofa

preformed CsCl

density

gradient (10).

By

applying

this

technique

and increasing the resolution of

density

in sucrose

and

CsCl

density

gradients,

itwas

clearly

dem-onstrated that there existed

heavy (H)

and

light

(L) infectious virions in almostall virus

prepa-rationstested.

Experiments to correlate

predominance

of

eitherof the two

populations

withthermal

in-activation led to the conclusion that H

might

representan intermediate state between

infec-tious and noninfecinfec-tious virions.

Furthermore,

essentially

the same

density

shiftwasdetected

after treatment of HSV with

limiting

dilutions

ofantiserum.

Also,

statesofHSV sensitizedwith

737

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738 YANAGI

earlyimmunoglobulinG (IgG) (mostly

comple-ment-requiringneutralizing IgG)and HSV

neu-tralized with early IgG plus complementwere

clearlydifferentiatedbybuoyantdensity analy-ses. Thus, acorrelation between the change in

physical characteristics of HSV particles and

loss of infectivity was established. Details of

these experiments are presented anddiscussed

in this report.

MATERIALS AND METHODS

Virus and cells. HSV type 1 (HSV-1) strainHF, HSV type 2 (HSV-2) strains UW-268 and NO (23), and African green monkeykidney (Vero) cellswere maintained inthislaboratory.HSV-1 strainMaclntyre and hamsterkidney (BHK-21/C13) cells have been described (27). Cells were grown in Eagle minimal essentialmediumsupplementedwith 10% calfserum at370CinaC02incubator. 3H-labeled HSVparticles werepreparedbyinfectingcells with5PFU ofHSV

per cell at 370C andlabelingthe HSV-infected cell

cultures with 5

ACi

of[3H]thymidine (5.0 Ci/mmol; NewEngland NuclearCorp.,Boston, Mass.)perml in minimal essential mediumsupplementedwith 1%calf serumfrom4to20hpostinfection. The labeled virions were harvested at the end of the labeling period.

Approximately50% of3H-labeled virionswerepresent

in theculture medium. Freeornon-cell-bound virus preparations were made by centrifuging the labeled culture fluidat 2,000 rpmfor 15min toremove cell debris. Forpreparationofcell-boundvirions,infected cells were scraped off witha rubberpoliceman and suspended infresh maintenance medium of thesame volumeasthe culture fluid,frozen and thawed three times,using acetone-dry ice, andcentrifugedat2,000 rpm for15mintoremovecell debris.Specific infectiv-ity ranged from50to 150PFU/dpm.Fresh free virions wereused foranalyses unlessotherwise mentioned.

Plaque assay andradioactivitymeasurement. Titrations were done with primary chicken embryo cells.Preparation ofprimarychickenembryocellsand theHSVplaque assay procedure followedour stand-ardmethod (24). The radioactivity of fractions con-taining [3H]thymidine-labeled HSV particles were measured with the scintillation cocktail Aquasol 2 (New England Nuclear Corp.). Fractions containing radioactive materials which weresoluble in trichloro-acetic acid were precipitated onto glass fiber filters

(WhatmanGF/C)withanequal volume of cold 10%

trichloroacetic acid-0.04 M sodium pyrophosphate andcounted withAquasol 2.

Buoyantdensity determination. To determine buoyant densities of HSV particles in sucrosesolution, alinear30to70%(wt/vol) sucrose gradient was made inEarle balanced salt solutioncontaining 0.5% lactal-buminhydrolysate and 10 mM

N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic

acid (HEPES) buffer,

pH7.6.Centrifugationwascarried out at 25,000 rpm

for 16 h at 20C, using a Beckman SW27.1 rotor. Densities of fractions weredetermined by measure-mentoftherefractive indices at

200C,

using aBausch & Lombrefractometer.Fordeterminationofbuoyant densities in CsCl solution, a linear 10 to 30% (wt/wt) CsCl solution containing virions was gentlyformedon the 36% (wt/wt) CsCl solution containing HSV,

ac-cording

toLawrence's method(10).Centrifugationwas

performed at 15,000 rpm and

00C

for 15h, using a

Beckman SW41rotor.Therefractive indices of frac-tionsweredeterminedat

250C.

Percoll,apreparation

of colloidal silica coated with polyvinylpyrrolidone,

was purchasedfrom Pharmacia (Uppsala, Sweden). Anisotonic Percollsuspensionin 10 mM Tris-hydro-chloridebuffer,pH 7.6,wasmadebyaddingsucrose to0.25 M.CentrifugationinthePercollsuspension, of which thestarting densitywasadjustedto1.080g/ml,

wasperformedat25,000rpm for 50minat00Cin a

Beckman type 65 rotor. Densities of fractionswere

determinedbymeasurementoftherefractive indices

at200C.

Ratesedimentationanalysis.Alinear 10 to 30%

(wt/wt)sucrosegradientwasformedonacushion of 70% (wt/wt) sucrose solution in Earlebalanced salt solution containing lactalbumin hydrolysate and 10 mMHEPES, pH7.6.Sedimentationwascarried out at20CinaBeckman SW41rotorforanalysesofHSV

particles. 32P-labeledsimian virus40,whichwasused

as asedimentation coefficientmarker,was agiftfrom N.YamaguchiofourInstitute.

Antiserum, IgG, and complement.

Hyperim-munerabbitantiserum and rabbitearly IgGtostrain HF weredescribed previously (28).Theneutralizing potenciesof theantiserum andIgG,determinedbya

plaquereductiontest,were1:640 and1:5,respectively,

withoutcomplement.Thesourceofcomplementwas unheatednormalguinea pig serafree ofnonspecific

inhibitors. Hemolytic units were determinedby the

procedure previously described (25), and a dilution

containing 10 U/0.1 ml was used. Experiments of neutralization withantiserum,IgG, andcomplement weredoneaccordingto ourstandardprocedures(28).

Inbrief,0.20 ml of virus and 0.20 ml of theindicated

dilution ofserum orimmunoglobulinweremixed and incubatedat370Cfor1h and thenchilledonice and

immediately subjectedtodensity analysis. A mixture

replacing serum or IgG with diluent served as the

control. Thediluent forneutralization without

com-plementwasminimal essential medium supplemented

with 2% calf serum and that for sensitization and

followingcomplement treatment was physiological

sa-linecontaining0.001M MgCl2-0.0025M

Tris-hydro-chloride buffer (pH 7.8)-0.5% gelatin (Difco

Labora-tories, Detroit,Mich.).

DNase I treatment. DNase I(grade I) frombovine pancreaswaspurchasedfrom BoehringerMannheim

GmbH(Mannheim,WestGermany). Enzymatic

diges-tionwasdoneat300Cinareaction mixture containing DNase Iat afinalconcentration of 200 or 400,ug/ml and 5mMMgSO4in Earlebalanced salt solution. At the end of thereaction, an equal volume of cold 10% trichloroacetic acid solution containing 0.04 M sodium pyrophosphate was added to the reaction mixture,

which waskept on ice for 10 min and then spun at

2,500 rpmand50C for20min. The supernatant was

removed, the precipitate was washed with cold 5%

trichloroacetic acid containing 0.04 M sodium pyro-phosphateandrecentrifuged, and the wash was pooled with the supernatant. Theradioactivities of the super-natantandprecipitateweremeasured by using Aqua-sol2(New England Nuclear Corp.).

Electron microscopy. Virus suspensions were concentrated by centrifugation at 15,000 rpm for 90 J. VIROL.

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min.Negatively stained whole mounts were prepared by touching a Formvar-carbon-coated grid to a virus suspension and touching the grid to 2% phosphotungs-ticacid, pH 7.3. Excess stain was removed with filter paper. Anelectronmicroscope, JEM-100 CX (Japan Electron Optics Co., Tokyo), was used at a magnifi-cation ofx20,000 toX33,000.

Otheranalyses. Ultrasonication was carried out

inice-cold water; 15-ssonication was repeated at 15-s intervals, using a Rapidis A 180 G ultrasonicator (Ul-trasonics Ltd., Shipley, York, England). HSV DNA wasextracted andcentrifuged in a CsCldensity gra-dient to determine the buoyant density, as described previously (26). UVirradiation was done with a rock-ing UV irradiator (Tominaga Ltd., Tokyo). Trypsin was aproduct of DifcoLaboratories.

RESULTS

Presenceoftwopopulations of infectious HSVparticles with different buoyant

den-sities and spontaneous or thermal transi-tion between thetwostates.We first triedto useequilibriumsucrosedensity gradient centrif-ugationtoanalyze the buoyant density charac-teristics of highlyinfectious HSVparticles,

be-causesucroseis knownto bemuch less

harmful

totheinfectivity of HSV than

CsCl

(20).It was

learned thata 30 to70% sucrosedensitygradient

gavesatisfactory density resolution,with an

in-fectivity recovery rate of >80%. Most of the presentexperiments used

non-cell-bound

virions ofHFstrain HSV-1 from infected BHK-21/C13 cells or Vero cells. It was revealed that the buoyantdensity insucrosesolutionwasbimodal rather thanhomogeneous withmostvirus

prep-arations. Thebuoyant densities of thetwobands

were

determined

tobe1.207and1.226

g/ml

with

virus from Verocells. The ratio ofvirus

quanti-ties between the formerand latter bands varied somewhat fromoneviral

preparation

toanother.

A

typical

example

is

depicted

in

Fig.

1A.

(The

heavy and light bands will bereferredto as H

andL,

respectively, hereafter.)

The ratio of

in-fectivityto

radioactivity

of

[3H]thymidine

incor-porated

intoHSV DNA

(specific infectivity)

was

almost

equal

between H and L. It should be noted that both H and L bands were

always

sharp and

reproduced

theindicated

density

val-ues in

repeated

tests, and that there were no

other

detectable

populations

internediate

be-tween or outside the two

populations.

Cell-bound virions released from infected cells by freeze-thawinggave thesameresultsasdid

non-cell-boundvirions. Lwas

recentrifuged

after di-alysisagainstEarle balanced salt solution

con-taining 10 mM HEPES

buffer,

pH 7.6,

at

0°C

overnight; the bimodal viral distribution was

again shown, but thesametreatmentof H left

littleL (Fig. 2).TheresultssuggestthatL

con-vertedto H but that the reverse did not take

place.

Conditions favoring the L to H conversion

were studied further. When the virus in the

mediumwasincubatedat37°C for 6 h, Hseemed

0

x

0. 0i

+1

x

1

aI4

I?

0

0-I

0'

C?

0

fraction no

FIG. 1. Buoyantdensityanalysisof

HSVparticles

by equilibrium centrifugation in a sucrose density

gradient.

[3H]thymidine-labeled

HSV-1 particles

(HF strain) were propagated in BHK-21 cells. A harvestedculture mediumcontainingnon-cell-bound

HSVwascentrifuged at a lowspeedtoremovecell

debris, treated as indicated below, loaded onto a linear 30 to 70%

(wt/vol)

sucrose densitygradient,

andcentrifugedat25,000 rpm and2°Cfor16h ina

Beckman SW27.1 rotor. Theradioactivity of300

Al1

outof each500-,ul

fraction

wasmeasured and

plotted.

(A)

Virusnotpreheated. (B) Virusincubatedat37°C

for 6h before equilibrium centrifugation. (C) Virus

preincubatedat37°Cfor20h.Symbols: bars,

infec-tivity;

0,

radioactivity;

0,

density.

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H conversion from L toHconcomitant with loss of

1.5 A infectivity was

encountered.

A reverse

transi--126tion,

from H to

L,

was never

observed.

__

9124

Distinction of two populations of

infec-volO0 \ 122 tious HSV virions with different

buoyant

densities

in a

CsCl

gradient.

When we

ap-z1.200

plied

Lawrence's

improved

"flotation up"

0.5\\

1.18 method to HSVbandinginCsCl,wefound that

\.5

\

1.16

therecovery of

infectivity

was asmuch as

25%,

incontrast tothedrasticreduction of

infectivity

in the

conventional

banding using a non-pre-formed

CsCl

density

gradient.

026

- A

major

feature of the virusdistribution

pat-v0

1.0 B

1 1.24 ;

-

tern

in

CsCl

(Fig.

3A)

was

the

formation

of

two

bands, as was the case in the

~~~~~~~~~~1.22~

a. \ \ 122

,,,density

gradient. Again,

both of these bands

as Yt

1.20

t contained infectious HSV virions, and the

spe-1.18

cific

infectivities of the two

peaks

were almost

equal.

Itwas

noteworthy,

however,

thatH

pre-C

o126

,,

| C

2

X

6122

A

x0

'0

20 tl

tlth\

to

E.30 frationno

It20

lov

1 1l 1 1 1ld

<

E

a.

R

t0

suroe

gadint.

2 E

Equilibrium2entrifuga-.0. _5o

10O 20C o

traction no'. 2

FIG. 2. Recentrifugation of isolated H and L bands insucrosegradients.Equilibrium

centrifuga-tion insucrose density gradients was done asgiven in thelegend to Fig. 1. (A) Buoyant density distribu- 3

tion of[3H]thymidine-labeled HF particles, which B \ wereproduced from Vero cells. (B) Fractions 13 to 15

in(A)thatcontained mostly L were pooled, dialyzed

30

against Earle balanced salt solution at 00C, and 'o2 E10

recentrifugedtoequilibrium in a sucrose densitygra- x

dient. (C) Fractions 8 to 10 in (A) that contained

mostly H werepooled, dialyzed as above, and recen- a. ?

1.25

u

C

trifuged. Symbols are the same as in

Fig.)1.

i

.l

\5

c,

toincrease atthe expense of L (Fig. lB). This f c

was

repeated

with another virus

preparation

originally containing little H, in which the H 5 10 15

20

25 30 35

peak was newly apparent after incubation at fraction no.

370C. The L to H conversion also took place FIG. 3. Buoyant density analysis of HSV by

equi-when L virionswere left standing at50Cfor 18 librium centrifugation in aCsCIdensity gradient. To

days, although specific infectivity was reduced a suspension ofHSV-1 virions (HF strain) labeled

toabout1/500 inthis case.When incubation of with

[3H]thymidine

wasaddedaconcentratedCsCI

the virus suspension at370C was prolonged to solution up to36%

(wt/wt).

This wasplacedat the

20h, a markeddecreasein L witha concomitant bottom ofacentrifugetube,onwhichalinear 10to

increase in H was seen (Fig. 1). The latter

30%

(wt/wt)

CsCl

gradient was gently formedat 40C.

inactivatedparticls

fEquilibrium

centrifugation wasperformedat

15,000

diactivated

particles

formedabandat thesame rpm and

0°C

for 15 h inaBeckmanSW41rotor.(A)

densityas didinfectiousHobservedinthe pre- Virus not pretreated. (B) Virus incubated at450C for

cedingexperiments.When the virus was heated I h before centrifugation. Symbols are the same as in

at450Cfor 1 h instead ofat370C,anearly total Fig.1.

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dominated in CsCl solution in contrast to the

predominanceof L insucrose.Thissuggeststhat

CsClandexposureto370C exerted similar

influ-ence onthe virions. When the viruswasheated

at450Cfor 1h, onlyonepeak with little

infec-tivity wasobservedat adensity corresponding

to the Hband (Fig. 3B). The disappearance of

theinfectious Lbandwasnotdueto

disintegra-tion ofvirions, but was solely due to its total

conversiontoH, becausetherecoveryof

radio-activity of 3H-labeled viral DNAwas>90%.

Bimodalbuoyant

density

patternof HSV

ofdifferenttypes,strains, andceli origins.

Whether the results described above could be

reproduced with other strains of HSVor with

HSVof different celloriginswasnextexplored.

The virus types, strains, and cell origins are

listed in Table1.The resultswereuniform with

respect tothebimodalpatternofviriondensity,

although in thecasesofthe HF strain from Vero

cells, the UW-268 strain from BHK cells, and

the NO strain from BHK cells, L was much

smaller inproportiontoHinCsClsolution than

inthe othercases.

Evidencethat H isnotanartefact. A series

ofexperiments was carried out to resolve the

question of what caused the occurrence ofthe

two HSV populations with different buoyant

densities. First, theisopycniccentrifugation

pat-ternsin asucrose density gradient of free and

cell-bound HSVparticleswerecompared.

Essen-tially, thesamebuoyantdensity characteristics

as described above were observed with both

virions. Second, it was suspected that virions

with different densities resulted fromanosmotic

pressure exerted on HSV particles in sucrose

and CsCl solutions or from permeation of the

solutes intotheviral particle.Therefore, Percoll

(colloidal silica coated with

polyvinylpyrroli-done)waschosentotestthese possibilities.

Per-coll hasa verylow osmolarity andis unable to

passbiologicalmembranes. This coatedcolloidal

silica is compatible with the integrity of living

cells (16) and of virions (15). There appeared

clearly two populations of HSV virions in the

Percolldensity gradientatdensities of1.072and

1.054 g/ml,theinfectivityrecoverybeing >90%.

Third, whetherornotaggregation of HSV

par-ticles whichmightoccurduring the

centrifuga-tion contributed to the appearance of the H

bandwasquestioned, although the nearly

iden-tical specific infectivities of the two bands

seemed to disprove this possibility. We first

testedwhether thedispersalof HSVparticles by

ultrasonication couldconvertHtoL.

Ultrason-ication for 30or 360sof HSVsuspensions

con-taining both H and L did not show such a

change, but ratherappearedtoconvertL toH,

probablyduetoadegradationeffect of

ultrason-ication (Fig. 4). Next,L andH were each

sub-jectedtoaratesedimentationanalysisina10 to 30%sucrosegradient (Fig. 5).AnHSV

prepara-tioncontaining L almost exclusively andits

heat-denatured sample consisting exclusively of H

werecompared. They each formedone

symmet-ricpeakatnearlythesame distancerelative to

a marker, 3P-labeled simian virus 40 virions

(240S), thesedimentation coefficient being

ap-proximately 1,000 S (although H appeared to

sedimentslightlyfaster thanL). Thus,

aggrega-tion of HSVparticleswasnot thereasonforthe

appearanceof H. This conclusion has been sup-TABLE 1. Buoyant densities insucroseandCsCI solutionsof HSV-1 and HSV-2 virions

Virus Buoyantdensity(g/il)b

Sucrose CsC1

Type Strain Hostcell

HC L H L

I HF BHK 1.226 1.206 1.292 1.253

I HF Vero 1.227 1.205 1.288 1.250

I MacIntyre BHK 1.229 1.208 1.301 1.255

I MacIntyre Vero 1.228 1.206 1.287 1.245

II UW BHK 1.232 1.211 1.283 1.250

II UW Vero 1.235 1.210 1.292 1.253

II NO BHK 1.230 1.210 1.286 d

II NO Vero 1.231 1.209 1.290 1.250

aBHK-21andVerocellswereinfected with each strain of HSV-1and HSV-2atamultiplicityof 5PFU/cell

andwere incubated at 370Cfor 20h. HSVparticles wereradiolabeled with [3H]thymidine from 4 to 20h

postinfectionandappliedtobuoyantdensityanalyses by equilibriumcentrifugation insucroseand CsCldensity

gradients.

bThebuoyantdensityvalues represent averages from three ormoreexperiments.Thebuoyant density of HSVinfectivity andradioactivitypeaksweredeterminedbymeasurementof refractive indices.

'H, Heavyvirions inthe lowerband; L, lightvirions in theupperband.

d

_,

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digested

with 0.25%

trypsin

at

370C

for 15

min,

the buoyant density decreased from 1.205 to

2

I 1.189

g/ml.

The titerwasreduced from 1.8x107

x2 t L ar to 1.2 x

103 PFU/ml

after this

enzymatic

treat-ment.Aninvolvement of protease in theL toH

o

1200$

conversionwasthus

unlikely.

1 9\ 1I Theresultsdescribed in this section indicated

I that the presence of H and L populations in

4_1150 HSV

preparations

was not an

artefact,

implying

instead that there are two different

physical

B

05mn.

t250

states of HSV particles.

Effect ofUVtreatment.The effectonHSV

02 E of

irradiation

with a low dose of UV light was

°

2- f

N12

examined.

When HF virions from BHK

cells

xX'g were irradiated under a 15-W UV

lamp

at a

distance of 10 cm for 20 s, the

infectivity

de-1 ( \ [1150| creased from 3 x

106

to 1.1 x 104 PFU/ml, but

1.150 the viralbuoyantdensity didnot

change.

Electronmicroscopic observations.

Elec-tronmicroscopic observations werecarriedout

C 6min

-1250

15A

2~~~~~~~~2O

240S T

71.150 2v

a.

10 20 30 0.5

f raction no IO5-.

FIG. 4. Ultrasonication treatment ofHSV L and C'J

Hparticles.

Equilibriumcentrifugation ofHFvirions 0i frominfected BHK cells insucrosedensitygradients

wasperformedasinthelegendtoFig.1.(A) Virions B

2405

notultrasonicated. (B) Virions ultrasonicatedfor30

OI

s. (C) Virions ultrasonicatedfor360s. Symbolsare _le

thesameasinFig.1. lo- -2x

0.~~~~~~t0

ported

further

by

anelectron

microscopic

obser-vation that showednoaggregates ofHSV par- 0 1 X

ticles under our

experimental

conditions.

<,I

X

Fourth, DNAcomponents ofHandLparticles

werecompared,sinceitwasreported that HEp-

''

2cells infected with HSVgaveriseto twokinds

ofprogeny

virions

which couldbeseparated by fraction no.

rate zonal

centrifugation

inlinear sucrose gra- FIG. 5. Rate sedimentation analysis ofHSVH and

dients,and the morerapidly sedinentingvirions L particles. HSV-1 (HF strain) was propagated in

contained DNA of three different

densities

(1). BHK-21 cells, and loaded onto a linear 10 to 30%

The buoyantdensities of DNA extracted from sucrosedensity gradient with a 70% sucrose cushion the isolated H and Lvirions shown in Fig. 1A atthe bottomofthetube,andspunat20,000 rpmand

were both 1.725g/ml without any minor com-

2°C

for35mininaBeckmanSW41rotor.

32P-labeled

ponentsorcontaminants (Fig. 6). This result as simian virus 40 virions, with asedimentation

con-well as the above rate sedimentation analysis stant of 240S, were included in the same tubes as a

deniedattachmnof

nsedimentation

coefficientmarker. The radioactivity

deniled.

attachmenttof

nonviral

DNA

toat

HSV of250,u of each370-,ul

fraction

was

determined

and

particles. Finally, the possibility that an un- plotted.

(A)

HSV preparation containing an L

popu-knownproteaseactioncaused the buoyant den- lation almost exclusively. (B) Hpopulation prepared

sity changeofHSVwas tested.When HFvirions by preincubation of the HSVpreparation at 37°C for

from Vero

cells

composed mostly of L were 24 h.Symbolsarethe same as inFig.1.

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CHANGE OF HSV PHYSICAL STATE AND INACTIVATION 743

fraction no.

FIG. 6. CsCldensity gradientequilibrium

centrif-ugation analysis of DNA from H and Lparticles.

HSVDNAswereextractedfromseparatedHandL peakswhich hadbeen fractionatedbysucrosedensity gradient equilibrium centrifugation of

[3H]thymi-dine-labeled HFstrain virions(see Fig.1A).(A)DNA from Lpeak. (B) DNA fromH peak.Symbolsarethe same asin Fig.1.

to find possible morphological differences

be-tweenLandHvirions. Alarge portion ofboth

L and H virionswasfoundtoallowpenetration

ofphosphotungstic acid. Itwasconcluded,

there-fore, that separation of Land Hpopulationsin

adensitygradientwasnotdue toadifference in

integrity of the viral envelope. Although the

rupture oftheenvelope and the resulting

pene-tration of the solutes in the gradient solution

should cause some difference in the virion's

buoyantdensity theoretically,it does notappear

tocause asbigadensity changeasthatoccurring

in the LtoH transition.Itwas analteration of

theoutershapeofavirion that characterized H

particles.Most of the H virions hadan

extraor-dinarily extended part which had a panhandle

orsmall bulblikestructure(Fig. 7). Althoughwe

observedsimilarparticlesofanalteredshapein

the L-virionpreparationalso,their numberwas

so small that they were probably H particles

newlyappearingduringthepreparationofgrids

for electronmicroscopicobservation. Itwas

ob-served that crude virus preparations before a

high-speed centrifugation also contained virus

particles which had such extended parts. It is

notclearatthe moment,however,whetherthere

isanydifferencebetween L and Hvirionsin the

volume ofparticles.

Effect of antiserum treatment. The effect

ofantiserum treatment on the buoyant density

ofHSVwasstudied incomparisonwith thatby thermal treatment. The HF virions used in this

analysiswere produced in Vero cells. A hyper-immune rabbit antiserum with a titer of 1:640

was diluted to 1:640 and 1:1,280. A virus sample wasmixed withan equal volume of thediluted antiserum andincubated at 37°C for 1 h. The

reactionmixturewaschilledonice at the endof

the reaction time and immediately applied to

sucrose density gradients and centrifuged for

buoyant density andsedimentation rate

analy-ses.Treatment with theantiserumatadilution

of1:1,280,whichreduced the infectivityfrom2.0

X 107to 1.14 x

107

PFU/ml(Fig. 8B), induced

adensity shift of the virions inafashion similar

tothe LtoH conversionbythermal treatment. The densities ofthe two viral bands after the

antiserumtreatment wereslightly heavier than the usualdensities ofLandH,being 1.210 and

approximnately1.23g/ml, respectively.When the

serumconcentrationwasdoubled, the infectivity

decreasedto 9.60 x 106PFU/ml, the proportion ofHbecamelarger, and the densities ofthe two

viral bands were 1.236 and approximately 1.21

g/ml (Fig. 8A).Asedimentationrateanalysisof

theantiserum-treated HSV showed thatnoviral

aggregates wereformedunder the experimental

conditions (Fig.8FandG). Tenfold-diluted

nor-mal rabbit serum did not affect the buoyant

density of HF virions (Fig. 8D and E).

Effects of sensitization with early IgG

andsubsequent neutralization by addition of complement. The density values of

anti-body-treated viruswere nothighly reproducible but fluctuated among repeated experiments, suggesting that subtle changesin experimental conditions influenced that result. One such

fac-tormight beserumconstituentsother than

an-tibody. Hence, the next experiment used IgG from an

early

antiserum which containedmuch

complement-requiring

neutralizing antibody.

In-cubation of HSV with early IgG diluted 1:5 at

37°C for1h reduced theinfectivity onlyslightly, i.e., from 6.6 x 106 to 2.6 x 106

PFU/ml.

The buoyant density ofHSV shifted to 1.209 g/ml

after this sensitization(Fig.9B). When

comple-ment wasadded tothe sensitized virusand the

viruswasincubated furtherat

37°C

for 30min,

the infectivity decreased to 4.6 x 102

PFU/ml

and a drasticdensityshiftto 1.246g/mloccurred

(Fig. 9A). The sensitized virus before

comple-ment addition hada

slightly

faster

sedimenta-tion rate than the nonsensitized control virus

(Fig. 9H and I), suggesting that

although

no

aggregation resulted under the

experimental

conditions, faster

sedimenting

virion-IgG

com-plexes were formed. The complement-treated

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744 YANAGI

A

B

-FIG. 7. Electronmicrographsof negatively stained H and L virus particles. (A) Hparticles. (B) Lparticles. Bars,500nmeach.

virions sedimented muchfaster, suggesting oc-currence ofvirus-IgG-complement aggregation

or some drastic structural change in the virus

particle (Fig. 9G).Treatment ofHSV with

com-plementinthe absence ofIgGdid notaffect the

viralbuoyant density (Fig. 9E and F). For

com-parison, thebuoyantdensitypattern of HF

nu-cleocapsids,whichwereprepared byNonidet

P-40 treatment (6), is depicted in Fig. 9D. The

buoyant densityof the nucleocapsid was 1.260

g/ml heavier than that of the complement-treated virions.

Next, sensitivity toDNaseIof the sensitized

and complement-treated HSV particles was

studied, since itwasreported that neutralization

of some enveloped viruses with complement

treatment caused drastic immune virolysis in which viral genetic materials becamesensitive tonuclease(14, 17,22).The viruswastreated in

the same way as in the experiments described above (see Fig. 9). Theinfectivities of the

com-plement-neutralized, sensitized, and control

vi-russampleswere 1.2 x

103,

1.9 x

106,

and 8.2 x

106 PFU/ml, respectively. Equilibrium sucrose

density gradient centrifugationgavethese

sam-ples almostthe same density pattern as shown

inFig.9.Topfractionsofthe peaks were pooled

separately, dialyzed againstEarle balanced salt

solutiononice overnight, and tested for DNase

sensitivity. Novirolysis, asevidenced by an

in-creasedenzyme sensitivity, occurred even after

theaddition ofcomplement, although the result

did not exclude the possibility that damage of

the viral envelope occurred without rupturing

thenucleocapsid (Table 2).

DISCUSSION

There have been many reports which have

dealt with separation of virus into more than

onepopulation by isopycnic or rate zonal

cen-trifugation, but in most cases the separation

resultedfrom coexisting degradedor immature

noninfectiousparticles.Inthecaseof HSValso,

separation ofdisintegratedvirions(21)orvirions

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cV)

0

a-0)

*I'

cn)

so

x

a-c]

x

'

a-lo

x

ac

a-0

K

fraction no. truction nu.

FIG. 8. AntiserumtreatmentofHSV virions. HF virions from infected Vero cells were used. Equilibrium

centrifugationinsucrosedensitygradients (A-E)wereperformedasin thelegendtoFig.1.Asedimentation

rate analysis (F and G) wasdone with aBeckman SW41 rotor at 15,000 rpm and 2°C for50 min. The neutralization reactionwasdoneby addinganequal volumeofdilutedhyperimmuneornormal rabbitserum andsubsequent incubationat37°Cfor1hatpH 7.6.Attheendofthereactionthemixturewaschilledonice

andimmediately analyzedbysucrosedensitygradient centrifugation. Virions reacted with:(A) 1:640 diluted

hyperimmuneserum; (B) 1:1280dilutedhyperimmuneserum; (C) diluent[controlfor (A) and (B)]; (D) 1:10

diluted normal rabbitserum; (E)diluent[control for(D)]; (F) 1:320dilutedhyperimmune serum; (G)diluent [control for (F)]. Symbolsarethe same asinFig.1.

associated with nonviral DNA (1) have been

reported. Ourreport,however, differs fromthose

casesinthatwearedealingwithinfectious

par-ticles. The existence ofHand L virions of full

infectivity havebeen reported so far with only

twoparvoviruses,nonenveloped minute virus of

miceand H-1 virus (3,4,8).

The Hand L virions of HSVasdefinedinthis

study do not seem to be

artefacts,

and L was

showntoconverttoHundercertain

experimen-tal conditions. This conversion seemed to be

irreversible. Circumstances

favoring

natural

degradation of viral

infectivity

tended to in-creasethe H

portion.

In

addition,

a

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746 YANAGI

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I01A Wda -O-- Ix Wda O

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TABLE 2. Resistance to DNase IofHSV treated with early IgG(sensitizedvirus) or with IgG plus

complement(neutralizedvirus)'

DNase Reac- Counts(dpm)' Virus or DNAprepnb I concn

tion

(u.g/ml)

tixme

Insolu- Solu-Virus treated with 0 0 594 24

early IgG plus com- 200 20 512 11

plement 300 180 562 4

Virustreated with 0 0 441 29

early IgG 200 20 428 29

300 180 440 40

Untreated virus 0 0 397 37

200 20 331 22

300 180 374 35

Viral DNA 0 0 14,690 120

100 1 585 13,968 100 7 78 15,499 aDNase Idigestionwasdoneat30°Cinareaction mixture containing 5 mM

Mg2e

in Earle balanced salt solution. [3H]thymidine-labeledvirionsorviral DNAswereused.

'HSV-1 (HFstrain) virions were sensitized with rabbit early IgG at37°Cfor1h,and thentreated with10U of crude complementpreparationat370Cfor 30 min. Control viruswas incubated with the reaction buffer withoutIgGand comple-ment. The infectivity titers of the control, sensitized and neutralized viruseswere6.6 x 106, 2.6 x 106, and4.6 x 102 PFU/ml,respectively.HSV DNAwasisolated from HF viri-ons.

'Radioactivityof3H-labeled HSV DNAinthepelletand supernatantafter trichloroaceticacidprecipitationand sepa-rationbycentrifugation.

ical difference between Land H

particles

was

demonstrated (Fig. 7). It might be presumed that the treatments of L particles resulted in such

morphological

changes as a result ofan

increasedfragility of the envelopeor tegument orboth. Ifadecrease in volume ofavirus

particle

accompaniesanalterationintheoutershape of the virus particle, it may cause an increasein

thebuoyantdensity.An

important point

emerg-ing from these results is that viral inactivation by thermal treatment as well as spontaneous

degradationare relatedto a changeinthe viral physical property, and even more interesting, thischangeseems toprecede loss of

infectivity.

Our

study

suggeststhatH mayrepresenta state

intermediate between infectious and

noninfec-tious virions. Inthisconnection,it is ofinterest

that the Hto Lratio varied dependingon the

virus strain and cell origin. Taking the above

presumption for granted, one would take this

fact assuggestingthepresence ofstrain-andcell

origin-dependent variances in the half-life of

viraldegradation.

Neutralizationof HF virions witha

sublimit-ing dilution ofantiserum causeda

density

shift

thatseemedtobe

basically

thesame asthe Lto

H conversion accompanyingnatural or thermal

degradation, although the absolutebuoyant

den-sity valuesof the two HSVpopulations became slightly higher after antibody treatment. This

slight increase indensity may reflect binding of

antibody molecules with the viral particle, be-cause we have determined the buoyant density

ofIgG,aswellas thatofIgM, in sucrose solution

to be 1.240 g/ml or greater. In fact, it was

re-portedthat antibody binding to the surface of

membranes effectivelyincreased the protein to

lipid ratios andcausedmembranes to band at a

higher density(7).

Aftercomplement treatment of early IgG-vi-ruscomplexes, the density increased from 1.209

to 1.246 g/ml, the sedimentation rate of the

complement-treated virions becoming much

higher. Thissuggeststhat aggregation of virions

mediated by complement occurred or that a

structuralchange in the virion-IgG complex was

inducedby complement treatment. It should be

pointedoutthatnoincrease insusceptibilityto

DNase wasdetected with the neutralized

viri-ons, in contrast to reports on

complement-me-diated immune

virolysis

of enveloped viruses suchas

equine

arteritis virus (17), type C virus

(14), andSindbis virus (22).Thestability of the

viralnucleocapsidstructure maydiffer fromone

enveloped virus to another, or the extent of

destruction of the viralstructure may depend on

experimental conditions. In any event, the

com-plement-requiring

neutralizing

antibody-sensi-tized virus may be analogous to the naturally

occurring H particles in that it represents an

intermediate state betweeninfectious and

non-infectious virions.

As to the possible correlation between the

physicochemical properties of a virus and its

inactivation by antibody, Mandelpresented the

hypothesis that neutralization of poliovirus

might be effected by attachment of a single

antibody molecule which irreversibly fixesthe

virustoonephysicalstate,whereas native virus has two interchangeable states detectable by isoelectricpointestimation(11). Atopographical change ofcoatproteininneutralized

picornavi-rusessupported this hypothesis (2, 9). Yoshino

and Isono (28) considered that a similar event

mightoccurin an

enveloped

virus suchasHSV.

The present result that

limiting

concentrations

of hyperimmune serum as well as early IgG consistingmostly ofcomplement-requiring

neu-tralizing antibody produced a change in HSV

densitysimilar to thenaturally occurringLtoH

conversion suggests the occurrence of a

struc-tural change in HSV. What changes actually

ensueafter sensitizationorneutralization

by

an-tibodyis not

clear,

and much isleft for future

studies. VOL.

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748 YANAGI

ACKNOWLEDGMENTS

IamindebtedtoK.Yoshino foradvice,support, and review of the manuscript,and to K.Hirosawa,A.Oda,and Y. Ohno forhelpinbeginningtheelectronmicroscopicobservations.

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