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0022-538X/78/0026-0435$02.00/0

Copyright© 1978 AmericanSocietyforMicrobiology Printed inU.S.A.

Intramembrane Changes Occurring During Maturation of

Herpes

Simplex

Virus

Type 1: Freeze-Fracture Study

MARCOS RODRIGUEZ* ANDMONIQUE DUBOIS-DALCQ

Infectious Diseases Branch, National Instituteof Neurological and Communicative Disorders and Stroke,

Bethesda,

Maryland

20014

Received for publication17November 1977

During thematuration oftwo strains ofherpessimplexvirus type 1 (VR3 and

Patton), intralnembranechangesweredetected with the freeze-fracturetechnique

in theviral

envelope

and theinfected cellplasma membrane, and these changes

werecompared with data obtained from thin sections. Regardless of the strain,

the innerleaflet of the viralenvelope of extracellularvirions wascharacterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and

plasma

membrane. Addition ofIMP, which probablyrepresent

virus-coded proteins, was detected in the viralenvelope only after budding from the nuclear membrane, whereasitoccurred during envelopment of capsids at

cyto-plasmic

vacuoles.

Fused membranes also showed one of their fracture faces

covered withahigh density of IMP similartothatof thematurevirionenvelope. Theinternal side of the membrane leaflet bearing thesenumerousparticles was

always characterized by the presence of an electron-dense material in thin

sections. Inaddition,the

plasma

membraneoffibroblastsandVerocellsshowed strain-specific changes: patches of closely packed IMP were observed with the VR3strain, whereasridges almost devoid ofIMPcharacterized theplasmalemma ofcells infected with the Patton strain. These intramembranechanges, however,

were notobserved as

early

as herpes membrane antigens. Thus, application of

thefreeze-fracture techniquetoherpessimplexvirus type1-infectedcells revealed

striking

structural differences between viral and uninfected cell membranes. These differencesare

probably

relatedtoinsertion and

clustering

ofvirus-coded proteins in the

hydrophobic

partof the membranebilayer.

Herpessimplex virustype 1(HSV-1) isaDNA ofthe membrane and similar tothat observed

virusand a memberof the Herpesvirus genus. ontheviral

envelope

(31).

The capsids

usually

acquire their envelope at Biochemical studies have

provided

evidence the inner nuclear membrane or sometimes at thatthe virus buds

through newly

synthesized

cytoplasmic membranes. The enveloped virion regions ofthenuclear membranecontaining

vi-containsatleast24

specific

proteins,

suggesting

mus-specific proteins

and

glycoproteins

(5). In

that

assembly

is a

complex

process (31). Once

addition,

new

antigens

thatareidenticaltothose

the capsid has been assembled in the nucleus, oftheviral

envelopes

have been detectedonthe

some membrane changes can be seen in thin plasma membrane of infected cells (24,25). To sections examined withtheelectronmicroscope explorewhetherthesebiochemicaleventsresult (6, 15, 18, 19, 21, 22, 28-32, 35).

Nucleocapsids

in structural changes in the membranes, the

are seencloseto

protruding regions

ofthe inner freeze-fracture

(FF) technique

was

applied

to

nuclear membrane which has a thin

layer

of HSV-1-infected cells. This

technique splits

cel-electron-dense material under its inner leaflet lular and viral membranesbetweenthetwo leaf-and will form the

envelope

of the virus. The lets and revealsintramembrane

particles (IMP)

enveloped

virus travels within a vacuole from

thought

to

correspond

to

proteins

and not

de-theperinuclearspacetothecellsurface, where tectable in thin sections (e.g., 3, 34). Various it is released into the extracellular space by stages of herpesvirus maturation were recog-reversephagocytosis (22, 28, 31). Theenvelope nized in FFreplicas,and thestructureofnuclear,

of the extracellularvirions showsathick layer

cytoplasmic,

and viral membraneswere exam-of electron-dense material on its inside and ined. Intramembrane alterationsweredetected

spikes onitssurface. Insome cell systems, the in several viral structures and

thought

to be

plasmalemma of infected cells has patches of relatedto theinsertion of virus-coded proteins

electron-dense fuzzapposedtothe innerleaflet inside the membrane. Inaddition,twodifferent 435

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sented at the annual meeting of the American hyde and 1% paraformaldehyde in 0.06 M cacodylate Society forMicrobiology,NewOrleans,La., 8-13 buffer at pH 7.2 (13). The latter fixative was then

May 1977.) replaced by a solution of 4% glutaraldehyde, and the

cells were detached from the culture surface with a MATERIALS AND METHODS rubber policeman. The cells were divided into two Virus and cells. The Patton (14) and McIntyre aliquots and spun down in fixative at 2,000 rpm for 30 (VR3) (16, 21) strains of HSV-1 were used in this min.Cells to beprocessed later for thin sectionswere study. Humanembryo foreskin fibroblasts were ob- pelletedinconicalcentrifuge tubes, whereas those for tained commercially from Flow Laboratories, Inc. the FF study were spun down onto acushion of agar. (Rockville, Md.). Vero cells were obtained from the Furthertreatment of thecellstoobtain thin sections American Tissue Type Collection (Rockville, Md.). and FFreplicas wasdescribed indetailpreviously (13). Cells were grown in a 5% CO2 atmosphere at 370C Briefly, cells to be thin sectioned were postfixed in until they were confluent. The growth media was osmic acid,dehydrated in alcohol, and embedded in Eagleminimum essential mediumsupplementedwith Epon.Standard and serial thin sections were cut with 10%heat-inactivatedfetal calf serum, 100 U of peni- adiamond knife and stained with uranyl acetate and cillin G, and 100

jig

ofstreptomycin sulfate per ml. lead citrate. Cells to befreeze-fractured were

equili-Both strains of HSV-1 were used after two passages in brated with 25%glycerol in water, frozen in Freon 22, humanfibroblasts. Pools of virus were obtained from stored in liquid nitrogen, and freeze-fractured at cells showing intense cytopathic effect. Cells were -1180Cin aBIzers360 Mapparatus. Electron micro-frozen andthawed three times. The fluidscontaining graphs were taken with a

Philips

201electron micro-released and cell-associated virus were clarified by scope. The measurements ofstructures observed in low-speed centrifugation and stored at -70°C. The thosemicrographswere done with aHewlett-Packard viral titers of these pools, as measured by plaque assay Digitizer.Student's t test was used for statistical anal-(11), were 3.5x 107 PFU/ml for the Patton strain and ysis.

2 x 106PFU/ml for the VR3 strain. Both viral pools Nomenclature. The nomenclature proposed by andcells were free ofmycoplasma as tested by meth- Branton et al. (7) was adopted to describe the changes odsdescribed elsewhere (27). observed in cellular membranes. Briefly, the mem-Infection of cellmonolayers. Stationary mono- brane leaflets in contact with thenucleoplasm or

cy-layersgrown in 75-cm2Falcon flasks(Falcon Plastics toplasm weredesignated P faces (fromprotoplasmic), DivisionofBioquest,Oxnard,Calif.)wereinfectedat whereas those closest to the perinuclear or extracel-amultiplicityof10PFU/cellin the case of the Patton lular space and the vacuolar lumen were designated E strain and0.3PFU/cell in the case of the VR3 strain. faces (fromexoplasmic).

The virus wasadsorbed to the cells for 1 h at 4°C on RESULTS

arocking machine and subsequently incubated for 30 Virus growthcurve.Usinga

multiplcity

of

minat37°C to allow viral penetration.

Vion

owth

ce.

U s a

synchrof

The nonabsorbed viruses wereremoved, and cells infectionof 10PFU/cell, all cells were synchro-wererefed with minimumessential medium containing

nously

infectedwith thePattonstrainofHSV-1

antibiotics and 2% fetal calfserum. Overlying fluids of (Fig. 1). After infection, the eclipse period of the cells infected with the Patton strainwere taken at 4, virus lasted approximately 8 h. Yield of infec-8, 12, 16, 21, 24,and 36 h postinfection (p.i.)to establish tious virus increased 3,500-fold between 8 and 21 aviralgrowth curve. These samples were titeredby h p.i. and thereafter remained stationary. the samemethods as the viral pools.

Immunofluorescent

staining.

Four to 8 h

Immunofluorescent staining. Cell surface and

Immunofluore

staining.

Fou of cytoplasmic-specific antigens were stained by indirect pi with the VR3 strain, approximately 10% of immunofluorescence techniques (20, 26) in cells in- the fibroblasts showed punctuate fluorescence fected from 4to48 h with the VR3 viralstrain. Human on their surface and diffuse staining of the

pro-hyperimmune serum with an anti-HSV-1 antibody toplasm. The proportion of these positive cells

titerof1:1,024 by indirect hemagglutinationtest (17) increased with time, and by 24 h p.i. all cells wasused and compared to human serawith no de- were positive. Controls were negative.

tectableherpesantibody.Anti-humanimmunoglobu- Electron microscopy. Observations made lin G coupled to fluorescein-isothiocynate was ob- with the FF technique on viral

morphology,

viral tainedcommercially (Progressive Laboratory, Balti- assembly,andcell membranechanges

occuring

more, Md.). during

HSV-1anfcll

were

angesucces-Electron microscopy. Viral maturation of two

dursig

and infection were analyzed

succes-strains ofHSV-1 was studiedin human fibroblasts. sively and comparedtodataobtainedfromthin

Thecells wereprepared for thin sectionand FF study sections.All ofthese results areschematized in atintervals of 4,8, 12, 16, 24, 36, and48 h p.i. with the lastfigure (seeFig. 16).

eachstrainused. Also, a few experiments weredone Viral morphology. In thin sections, typical

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1,064± 217, P<0.01) and than the density in IMP of the P faceoncontrolcell plasmalemma (2,815 ± 673 versus 1,021 ± 300, P<0.01). In

10

contrast,

the outer leaflet of the viral

envelope

did not show more IMP than the E face of

infected cell plasmalemma (Fig. 2). In

cross-frac-105 - / ture, the virions contained a circular

arrange-ment ofgranules of the size ofacapsid as

ob-/o served in thin sections(Fig. 2).

E

104

- °/ A number of virions did nothave the same

2_

/structural

featuresallovertheir

envelope.

In the

X-

/FF

replicas,IMPwereabsent on one pole of the

103/

innerhalf of the

envelope

ofsomeviruses

(Fig.

2and

3d,

andsee E in

Fig.

16).

The

density

of

IMP on these inner leaflets was 2,329 ± 870

102

IMP/,um2

whenareaswithout IMPwere

consid-10 °ered in the measurement. On the other

hand,

thedensity atthepoles covered with IMPwas

similarto that of virions withouta

pole.

Simi-10- larly, in thin sections, one region of the envelope

sometimes lacked the dense

fuzzy

material nor-I ,

mally

seen on the inside of the viral membrane

0 8 16 24 32 (18, 28) (Fig. 3a, and see E in Fig. 16). The

percentagesofvirons

showing heterogeneity

of HOURS POST-INFECTION their envelopes were closely similar both in FF

FiG.

1.

Growth

curve

showing

the

titers

ofreleased replicas and in thin sections and almost doubled

[image:3.509.48.238.52.320.2]

virusforthe Patton strain ofHSV-Iin humanfibro- between 12 and 48 h p.i. (Table 1). This suggests blasts.M{ultiplicity of infection =10PFU/cell. that some structural modification of the viral

membrane

progressively

developed

after the vi-herpesvirus (27) was first seen in the extracel- rus wasreleased from the cell. This modification lularspace at 8hp.i. with the Patton strain and of the virion

envelope

might be relatedto

deg-at 12hp.i. with the VR3 strain.Intheplatinum radatory changes relatedto aging of the virus.

replicas

obtained after

freeze-fracturing

infected Viral

assembly.

In thin

sections,

viral

cap-cellsat 12 to 48hp.i.,

hemispherical

membranes sidswerefirstseeninthe

nucleoplasm

at8h

p.i.

withmeandiameters of 164±12.7nm (standard with each viral strain used.

They

were either

deviation)

were observed in the extracellular scatteredoringroups, sometimes

forming

crys-space

(Fig. 2).

Theseconcaveandconvex mem- talline arrays

(27, 28, 31).

These

capsids

were

braneswere

identified, respectively,

astheouter

readily

identifiedin theFF

replicas

of cells in-and innerleaflets of free viruses because of their fected for8hor more

(Fig.

5) (23).Acluster of size and

shape

aswell astheir absence around granules

frequently

present inthecenterofthese uninfected control cells.

Histograms

of the di-

capsids

probably

corresponds

to the viralcore. ameters ofthese

hemispherical

structures and Protrusions ofthe inner nuclear membrane those ofvirionsobservedinthin sections showed overaviral

capsid

wereobservedinthinsections

asimilar distribution.

However,

the diameters of at 12 and 16 h

p.i.

in cells infected with the

virions that had been embedded in

Epon

were Patton strain and at 16 to 48 h

p.i.

in

cells

consistently

smaller than those that had been infected with the VR3 strain. In cells freeze-frozen in

glycerol

and fractured

(Fig. 4).

This fracturedatsimilar times

p.i.,

scattered

protru-difference in sizewas

probably

duetotheshrink- sions with a well defined circular base were ageof the virus

during

the

dehydration

process detected on the P face of the inner nuclear incells embedded for thinsectioning. membrane and hada meandiameter of170 nm A striking feature of theinner leaflet of the ±37.2

nrm

(Fig. 6, and see A inFig. 16). These viral envelope in the FF

replicas

was the in- protrusions were interpreted as viral budding

creased number of IMP per square micrometer. sites because of their size and shape and their The size of IMP on the virion was, however, absence in control nuclear membranes. Similar

similar to that of host cell IMP. The mean numbers ofelevationsonthe Eface of the outer

density of IMP persquare micrometer on the nuclear membrane were

slightly

biggerandhad inner leaflet wassignificantly higher than that less defined contours than those at the P face of the P face of the inner nuclear membrane (23) (Fig. 6). When the fracture plane jumped surrounding budding sites (2,815 ± 673versus from one nuclear membrane to another, it

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-e

r.

.,

.4

N

v

7

½~

w ~ ~--,~~~~ ,,, , ,

-~~~X

r

m

%,*

z

.x

FIG. 2. FFreplica ofvirions released in the extracellularspace (asterisk)at36hp.i.withtheVR3 strain. Thisand allsubsequentfiguresareofhumanfibroblasts. Thefracturefrequentlyrevealed theinnerhalf of theviralenvelopes (horizontal arrowheads)whichiscovered withIMP ingreaterdensitythan thatonthe P face oftheadjacentcell membrane. On theright, twovirions haveapoledevoidofIMP(short arrows). The outerhalf oftheviralenvelope (vertical arrowheads)hasasfewIMPasfragments ofthe Eface oftheplasma membraneofcellularfragments (E). Upper right corner,onevirion has beencross-fracturedand containsa circularstructureofthesizeofacapsid (long arrow)asobserved in thin sections(seeFig. 3a). x60/KiO.

FIG. 3. Details ofthe viralenvelope in thin sections (a) and FFreplicas (b andc). (a) Two virionsare shown whichhavedifferentcharacteristics. In theleftone,thecapsidisseparated from theenvelope byan electron-lucent space and an electron-densefuzz apposed to the innerside ofthe unit membrane which constitutestheenvelope.This dense material ispresentonlyatonepole ofthe virion shownontherightand is considerably thicker thanon thevirus shown ontheleft. Similarly, IMPcoverthe whole surface ofthe innerleafletshown in(b),whereastheyareclusteredatonepole ofthe virus shown in(c).X

150,XXJ.

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[image:4.509.81.465.69.543.2]
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VOL. 26, 1978

439

50

EJThin

Sections

8).

Much later in infection

(48

h

p.i.),

groupsof virionswere

occasionally

seenin the

perinuclear

F. F. Replicas

cisternae,

although nuclear budding sites were

40 rarely detected after 16 h p.i. (Fig. 9). Their

membrane was

strikingly

different from the

early

virions,

since

they

had much more IMP per square micrometer as did the

extracellular

30 >virion.Thissuggests thatsomevirionscanstay

z

z X fora

prolonged period

of

time

in the

perinuclear

3 X cisternaewhere theirenvelopecanprogressively

w 20 - _ enrichin IMP.

E_

EAt

12 and 16 h p.i. with the Patton strain,

serial sections revealedcomplete virions in

mem-10 _ brane-bound vacuoles localized at various

dis-tancesbetween the perinuclear cisternand the cell surface. Mostlikely, the membranelimiting

0 thesevacuoles isderived fromthe outernuclear

100 120 140 160 180 membrane, whichprobablyengulfsthe virus in

to to to to to a process similar to

phagocytosis

at the cell

120 140 160 180 200 surface

(Fig. 8) (28).

The inner leaflet ofvirions

DIAMETER OF VIRAL ENVELOPES (nm) lying inside vacuoles was rarely uncovered by

thefracture processsothatnoquantitative

ap-FIG.4Hsgs

o f

tiats

of

viral

ev

preciation

of the IMP

density

could be obtained

oeplicsobservedain)tinl

sections(oblique lines)nd

FF

for theextracellular virions. The inner leaflet of replicas (in black). Only virions which showed a

capsidin thin sections and rounded membranes that twovirionsobserved inside vacuoles at 12 hp.i. produced a shadowin FFreplicas were measured.

appeared

similarto that of virions detected in Bothhistogramshave a similarpattern, but adiffer- the

perinuclear

cisternaeatthattime.

ence of 20 nm existsbetween the mostfrequent di- Vacuoles connectedtotheplasma membranes

ameters. were identified inserial sections, and their

lim-iting membrane differed from theunopenones.

came clear that protrusions on both nuclear

Indeed,

at the site

where

a vacuole had

probably

membranes sometimes pertained to the same fused with the cell surface, the plasmalemma

budding

site

(Fig. 6).

These nuclear

budding

was often invaginated

into

vesicles similar

in

size

sites were

covered with

IMP

similar

in

size

and and shape to pinocytotic vesicles (Fig.

10a,

and density to those of the adjacent nuclear mem- see B3 in Fig. 16). These were

normally

scattered

brane.

all over the

cell

surface in uninfected

cells.

In

Changes in the distribution ofIMP on the contrast, when the E face of the plasma

mem-innernuclearmembrane wereobservedincells brane of infectedcellswas revealed by the

frac-infected withthe VR3strain andlateininfection ture, vesicles wereseen to

form

clusters around

(36 and48h

p.i.).

Roundareas

lacking

IMPwere depressions of theplasma membrane containing seenscatteredonthePface and measured 200 released virions (Fig.

lOb

and c). These mem-+ 12nmindiameter.

They

weresometimesseen brane infoldings might result from addition to

close to

budding

sites

(Fig. 6) (23).

In

addition,

the plasma membraneofthe limiting membrane

clusters of IMPondiscrete

protrusions

oftheP ofthe vacuolecontaining the virions.

face of the innernuclear membrane were seen Unenveloped capsids lying in the cytoplasm

occasionally

(Fig. 7).

These

protrusions

didnot and cytoplasmic envelopment were observed havethe size and

shape

of the muchmorefre- only late in infection(36 and 48 h

p.i.)

with both quent

budding

sites and

might

be related to strains of HSV-1. They were, however, more

discreteareasof

thickening

of the nuclear

mem-braneseenin thin sections

(29).

TABLE 1. Proportion ofvirions withheterogeneity

Individual

virionsreleased in the

perinuclear

in their

envelopes

at

different

times

after

viral

spacewereobservedatthesame timethat the infection

nuclear

budding

sites were identified in cells Virions (%) showing

infected with the Patton strain(12and 16 hp.i.). Type of virions heterogeneityat: In the

replicas,

the inner leaflet of the viral

envelope was characterized

by

a

population

of 12 h

p.i.

48 h

p.i.

IMPsimilartothat of thenuclear membraneat Virions with an excentric fuzz 34 66 the budding site (Fig. 8, and see B in Fig. 16). (thin sections)

These virions were often partly

engullfed

in a Virions with a pole devoid of IMP 38 58 depressionof theouternuclearmembrane

(Fig.

(FF replicas)

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[image:5.509.49.241.46.273.2] [image:5.509.252.445.553.656.2]
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VI.

3^I.t,^

~

N''''

r.~~~~~~~~~~~~I

.~ ~. .h~.~ -.

4 - -~~~~~~~40

[image:6.509.75.465.67.595.2]

~~~~~~~~~-,~~~~~~~~~~~~~Y

eiz-~f~J

FIG. 5. Portion of thenucleoplasm of an infectedcellthat wascross-fractured Viral capsids appear as granules arranged in a circle,sometimes enclosingagroup ofcentralgranules probablycorresponding to the viral core(arrows). x80,000~.

FIG. 6anld7. Aspect of nuclearmembranes at 16hp.i.with the Pattonstrain (Fig. 6) and at 36hp.i. with the VR3strain (Fig. 6 inset and Fig. 7). In Fig. 6 twoprotrusions are present on thePface of theinnernuclear membrane (P) and another on the E face of the outer nuclear membrane (E). The

number

ofIMP on the protrusions is similar to that of the adjacent nuclear membrane. The inset in Fig. 6 shows, at higher

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INTRAMEMBRANE HSV-1 441

ib

[image:7.509.60.445.68.285.2]

t

2~A;

J

S

:; '

;

FIG. 10. Interaction between extracellular virus and theplasmamembraneearlyafterreleasefromcells infectedfor12h with the Patton strainofHSV-1.Free viruses

sitting

in the extracellular space(onthe

right)

arein closecontactwithdepressionsoftheplasmamembrane in thin section(a)and FF(b). Vesicles,which areformedby plasmamembrane invagination, appearedtobeclustered aroundthesedepressions. This is moreclearlyseenwhenthe Efaceofsuchplasmamembranehas beenlargelyuncovered

during

the

fracture

process(c). The vesicles have beencross-fractured, and groupsof them, arrangedinageometricalpattern, areassociated withdepressionsprobablyrelatedtoextracellular viralparticles. Inone instance,a

cross-fracture

throughadepressionhasindeed uncoveredanextracellular virion (arrow). x60,000.

frequently observed in cells infected with the late in infection

(36

and48h

p.i.).

Fused

mem-VR3 strain. The virus was seen

budding

from

branes,

often

arranged

inaconcentricway,were

the

cytoplasm

into vacuoles which often con- observed in the

cytoplasm

ofinfected cells and tained

complete

virions

(Fig.

11

inset).

IntheFF consisted of

pairs

ofunit membranes whichare

replicas, elevations of various altitudes

protrud-

probably

extensions of the nuclear membrane ing into the vacuolar lumenwere identified as

(Fig. 12a) (6,

29,

30).

Pairs ofmembraneswere

budding sitesonthe vacuolarPface

(Fig. 11).

In

separated

by

anelectron-dense materialsimilar

contrast to the

budding

sites at the nuclear to the one observed atthe inner leaflet of the membrane, thePface of these

protrusions

was viral

envelope

(Fig. 13a).

These concentric fused covered witha

higher

density

ofIMPthan the membranes were

readily

identifiedin FF

repli-adjacent

vacuole membrane and resembled the cas.The fracture faces oftheleaflets

apposed

to

innerleaflet of extracellular virions

(Fig.

11,and the electron-dense material were covered with

seeC in

Fig. 16).

numerousand

closely packed

IMP ofa

density

Membrane

changes

not associated with similartothat of the innerleaflet ofextracellular viralmaturation. Membrane

changes

notas- virions

(3,078

± 783

IMP/,um2) (Fig.

12b and sociated with viral maturationwereallobserved

13b,

and see F in

Fig. 16).

Leaflets in contact

magnification,oneoftheseprotrusionsonthe PfaceontheleftofaflatareadevoidofIMP. Incontrasttothe elevations shown inFig.6,morediscreteprotrusionsseeninFig.7(arrows)arecoveredwith clustersofIMP. On theleft, thisclusteringis evendetectedon aflatareaofthe inner nuclear membrane(arrowhead). The protrusionontheright isshownathighermagnificationintheinset.Abbreviations:np,nuclearpores; cyt,

cytoplasm.Figure 6,x8O0,06;Fig.7,x60,000; insets, x120,000.

FIG. 8and9. Comparisonbetween virions within theperinuclearcisternaeat12(Fig. 8)and48h(Fig.9) p.i. with the PattonstrainofHSV-1.Figure8showsasinglevirion in which the innerleafletis covered with adensity ofIMP similartothatofthe Pfaceofthe inner nuclear membrane

surrounding

the

budding

sites (seeFig. 6).Incontrast, the innerleaflets ofthe virions showninFig.9arecovered withclosely packedIMP asseen ontheextracellular virions(seeFig. 2).Notethepresenceofnuclearcapsidsaligned alongthe inner nuclear membrane(arrowheads).Abbreviations:nuc,nucleoplasm;cyt,cytoplasm.Figure 8, x120,000; Fig. 9,

x80,OOO.

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4 I

I

p4v.

¾ 44

FIG. 11. Cytoplasmicenvelopmentat48hppi.withthePatton strainof

HSV-I1

Different stages of budding are detectedinthese three cytoplasmic vacuoles andcanbe compared withtheiraspect inthinsections (inset). InVl,protrusions interpretedas earlybuds show clusteringofIMPontheirPface (arrows); in V2, abud is starting to detachfromthe vacuole membrane andisprobablycompletely detachedinV3 (arrows). The inset showsunenvelopedcapsidslyinginthe cytoplasm anddifferentstages ofcytoplasmic envelopment (arrow-heads). x80,000.

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VOL. 26, 1978 INTRAMEMBRANE CHANGES OF HSV-1 443

~~~~~~~~~~~

5A.

Y.IS

FIG. 12 and 13. Aspectofconcentrically arrangedmembranes at 48 hp.i. with the VR3 strain in thin sections(Fig.12aand13a)andin FFreplica (Fig. 12b and13b). Themostexternal membrane isdirectly in contact with the cytoplasm and, inside this first concentric membrane, every other space between two membranes isseparated by an electron-dense material. After FF, the leaflet apposedto thefuzz in thin sections shows IMP inhigh densityas the innerleaflet of the extracellular virions (see Fig. 2).A central smootharea (arrowsin Fig. 12b and13b) might correspondto the absence of fuzz in someplaces ofthe concentriclayers (arrowinFig. 13a).Figure 12, x60,000; Fig.13,x150,000.

with theclear space inthin sections showed a distribution and density of IMP of the

mem-population

of

particles

similartothatof the E brane

surrounding

these

clusters, however,

did face of the nuclear membrane. not appear to differ from that of control cell

TheP face of the

plasma

membrane offibro- membrane. Thin sections ofidentical cells did blastsand Verocellsoccasionally showedstruc- notrevealchangesin structureandshapeofthe tural membrane changes that appeared to be membrane which could

correspond

tothe mod-strain

specific.

Indeed, theywereindependent of ifications detected in FFreplicas.

the host cell used since each strain produced DISCUSSION

identicalchangesinthetwotypes ofcells

stud-ied. The Patton straininduced, in 20% ofeach Thematuration ofHSV-1(strains Pattonand

cell type, the formation of ridges of various VR3) and thestructureof the infectedcell

mem-lengths,

175 to200nminwidth

(Fig.

14, andsee branes have been studied with theFF

technique

G in Fig. 16). These ridgeswerealmostdevoid and

compared

with data obtained from thin

ofIMP, but particles sometimes formed small sections. A distinction was made between early clusters at theperipheryof theridge.Theridges andlate events related to viral maturation and werecovered with fine filamentousmaterialrun- alterations of cellular membranes at sites where

ningalongthe axis.Cellsinfected with the VR3 no envelopment occurred (Fig. 16). The most strain did not show any ridge on theirplasma strikingobservation made in thisstudywasthat membrane Pface,but 20% of Verocellsand 5% the envelope of HSV-1 shows numerous IMP of fibroblasts had scattered areas of various after FF. Thus the intramembrane structure of

shapeand sizewhere IMP weredensely packed HSV-1, which assembles at nuclear and

cyto-together (Fig. 15, and see I in Fig. 16). The plasmicmembranes,isstrikinglydifferent from

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[image:9.509.59.449.66.365.2]
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.~~~~~~~~~~~~~~~.

[image:10.509.73.458.64.538.2]

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

W-INt,

FIG.~~~~~~

14.

and 15.

Coprsnbtenpamlma

ffbolssifce

o

8hwt

h

atnsri

7~,%

k- 4~~~~~~'.. 7

(Fig.

1s

fewer

IMPthn.hesurondngP

ac.heruracsowsfieilmetspaaleltothmjoaisofth :~~~~'~~~Ab~~~~A~~~~&,2 , -~~~~~~J:. o

FaiG.s

14ze

and

15sCmarisocn bedtwecen

plsatlemmas.hyaemd

fbolssiectelore48

of

hake

wIth.

the

PatontraFin.1

and 15, the rest of theplasmalemma showsaslightlyunevendistributionofIMP on its P face.

x100,X000.

that ofvarious enveloped RNA viruses (influ- observed at the site of virus budding as if the enza, Sindbis, visna, and vesicular stomatitis proteins of these viruses did not penetrate into viruses) budding from the plasma membranes the hydrophobic part of the bilayer. However, (1, 9, 10, 13, 33). In all of these cases, exclusion in measles infection, numerous IMP, smaller of IMPprobablyrepresenting host proteins was than most host cell IMP, have beenobserved at

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B2

ByB

B3®

FIG. 16. Thisdiagram summarizes FF observations on HSV-1-infected cells. The black dots apposed to cellularand viral membranes represent the intramembranousparticles (IMP) observed on the P face of cellular membranes and the innerleafletsof viral envelopes. Since nothing remarkable was observed on the outerleafletsof those membranes, they have been omitted from this scheme. The virus budding at the nuclear membrane(A)asweUasthe virion released in theperinuclear space(B)arecovered withasimilar number ofIMPasthe nuclear membrane. In contrast, virus budding incytoplasmic vacuoles (C) arecoveredwith IMP with adensity three timeshigherthan therestofthe vacuolar membrane. Virions enveloped at the nuclear membrane travelthroughthe cytoplasm within smooth vacuoles (Bl) and are released at the cell surfacebyaprocess ofreversephagocytosis (B2). The membrane around the released virus isfolded into vesicles(B3). Extracellular virions arecovered withthree times moreIMP than the nuclear and plasma membrane(D).Someofthese virions lack IMPat apole (E). Similarly, fused membranes (F) have fracture facescovered with three timesmoreIMP than the nuclear membrane. At the plasma membrane, ridges almost devoidofIMP(G) werespecific ofthe Pattonstrain,whereas clustersof IMP (I) were specific of the VR3 strain.

the sites of attachment of the nucleocapsid to ontheinnerleafletofthe viral envelope suggest

the modified

plasma

membraneaswellas on the thatviral proteinsaremore strongly anchored

viral buds(12). in the inner leaflet of the viral envelope and

Another remarkable membrane feature of most likely to theelectron-dense material seen

herpes infectionis thatthe numberof IMP per onthe inside of viral membrane.

square micrometer in the inner leaflet of the It has beenshown thatnewnuclearmembrane extracellular virion envelope, on the budding issynthesized during herpesinfection andthat

sites atcytoplasmic vacuoles, and onthefused the proteins used to build the nascent nuclear

membraneswasthreetimes higherthanthat of membrane are virus coded exclusively (4, 5). the host cellnuclearandplasmamembrane. In However, most of the nuclear membrane ap-contrast,thedensity ofIMP in theouter leaflet peared normal in FF replicas, whereas it did tag of the viralenvelopewas similar to that of the specificantibodies conjugatedtoferritininthin Efaceof the hostcellmembranes.Thinsections section studies (30). It is possible that viral

pro-revealedathick, electron-dense material under teins are evenly dispersed in the nuclear mem-the membranesbearingahigh density of IMP. branes of infectedcellsand thus not

distinguish-Thisobservation and the high density of IMP able from host cell proteins in FF replicas. In

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[image:11.509.93.405.61.362.2]
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IMPseemstooccurin theviral envelopeduring 1infectionisthat,in contrast to other budding thepassageof the virusthrough the cytoplasm, viruses, viral proteins in thematurevirioncan very early after release, or more rarely within beidentified inside the envelope bilayerasIMP the perinuclear cistern. It might be thatsome withacharacteristic density muchhigher than proteins that were integrated into the virion that of the nuclear and plasma membrane ofthe during nuclear envelopmentmoveprogressively host cell. Whenatechnique combining FF and fromanintemal

position

within theenvelopeor immunolabeling of intramembrane structures from the viroplasm toward the hydrophobic becomes available, it will be possible tobetter

layer of the viral envelope. Interestingly, viral define the nature of virus-induced

intramem-particles

isolated from thenucleus, which prob- brane changes. ably consist mostly of unenveloped capsids, have

been showntocontain viralglycoproteins similar

ACKNOWLEDGMENTS

tothose associated with the viralenvelope (4). We thank Jeff Brian Tate for hisexcellent photographical

The differencein structure ofthevirus-modi- assistance and Linda Dujack for herskillful thin sectioning. fledmembraneatnuclearand cytoplasmic bud- We also thank T. S. Reese andC. J.Gibbsforreviewingthe

fiedmmbraneatnulear ad cytplasmi bud- manuscript.

ding sites might be related to differences in M.R.and M.D.-D. are, respectively, VisitingFellow and nature between nuclear and cytoplasmic mem- VisitingScientist from the FogartyInternationalCenter.

branes. This was suggested earlier in

thin-sec-tionstudies onothermembers of the

Herpesvi-

LITERATURE

CITED

rus genus which revealed differences between 1. Bachi, T., W.Gerhard, J.Lindemann,and K. Muhl-envelopes ofvirions observed intheperinuclear ertahler. 1969.Morphogenesis ofinfluenza A virusin cistern and in the cytoplasm (18, 19, 25). The Ehrlichascites tumorcellsasrevealed by thin

section-presyoftwo strains of HSV-1' seems ingandfreeze-etching.J.Virol.4:769-776.

present studyoftwo strams ot H:rV-1 seems to 2.Benedetti, E.L.,I.Dunia, and A.Diawara.1973.The confirmthesedifferenceseventhough theywere organization of the plasma membrane inmammali seen

only

inFF

replicas.

Thedefinite structural cells. Eur. J.Cancer9:263-272.

characteristics

of the inner leaflet of the viral

3- Benedetti,

E. L.,

L-

Dunia, A. J. M. Vermorken, M. indeed

acquired

after

envelop-

Kibbelaar,

and H. Bloemendal. 1976. Aportraitof plasmamembranespecializationsineyelensepithelium ment at the nuclear membrane, whereas they andfibers. Biochim. Biophys.Acta457:353-384. were acquired during budding at cytoplasmic 4. Ben-Porat,T., and A. S.Kaplan. 1970.Synthesisof

membranes. It is

possible

thatviral

proteins

are proteins in cells infected with herpesvirus. V. Viral inserted

directly

atthesite of

envelopment.

The glycoproteins.Virology41:265-273.

5. Ben-Porat, T., and A. S. Kaplan. 1972. Studies on the fate of thehost cellproteinsinthatcaseisnot biogenesis ofherpesvirus envelope. Nature (London)

clear. 235:165-166.

Although the maturation steps of the two 6. Ben-Porat, T., and A. S. Kaplan. 1973.

Replication-strains of HSV-1 studied were

closely

similar,

Thebiochemical aspects, p. 164-216. In S. A. Kaplan (ed.),herpesviruses.Academic PressInc., New York.

differentstructures were seen in the P face of 7.Branton, D., S. Bullivant,N. B. Gilula, M. J.

Kar-the

plasmalemma

ofcellsinfected with different nowsky,H.Moor,K.Muhlethaler,D. H.Northcote, viral strains. The Patton strain induced

large

L.Packer, B. Satir, P. Satir, V. Speth, L. A. Staeh-ridges almost devoid ofIMP butcovered with lin, R. S.Steere, and R. S.Weinstein. 1975.

Freeze-etchingnomenclature. Science190:54-56.

filamentousmaterial. Thesefilaments

might

be 8.

Brightman,

N. W.,

L.

Prescott,

and T. S.Reese. 1974. attachedonthe inner side of themembraneface Intercellularjunctionsofspecialependyma,p. 146-165. (3) and be uncovered during the clearing of IMP InK. M.Knigge and D. E. Scott(ed.),Brain-endocrine observed on virus-induced ridges. The second interaction,

vol.

2.Theventricular system, 2nd

Inter-strainstudied(VR.) produced clusters ofIMP national Symposium,Shizuoka. S. KargerAG, Basel.

strain

studiedt (VRt3)

produced

clusters

of IMP

9. Brown, D. T., and B.

Riedel.

1977. Morphogenesis of which might containsome of thevirus-specific vesicular stomatitis virus: electronmicroscope obser-proteins identified earlier in the plasma mem- vations with freeze-fracture techniques. J. Virol. branesof infectedcells(24-26). However,fibro- 00.Brown,

21:601M609.

D.T., M. R.Waite, and E. R. Peeferkorn.

blasts infected with the VR3 strain manifested 1972. Morphology andmorphogenesisofSindbis virus membrane fluorescence much earlier and in as seen with freeze-etching techniques. J. Virol. many more

cells

than the membrane alterations 10:524-536.

detected byFF. Theclustersseenin FFreplicas 11. Cooper, P. D. 1967. The plaquep.243-311.In K.Maramoroschassay of animal viruses,and H.Koprowski(ed.),

resemble the aggregates of globular particles Methods in virology,vol.3. Academic Press Inc.,New seen at gap

junctions

between normal

ependy-

York.

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(13)

12. Dubois-Dalcq, M., and T. S. Reese. 1975. Structural proteins in theplasma membrane of infected cells. J. changes in the membrane of Vero cells infected with a Virol. 11:810-813.

paramyxovirus.J.CellBiol. 67:551-565. 25. Heine, J. W., P. G. Spear, and B. Roizman. 1972. 13.Dubois-Dalcq, M.,T.S.Reese,and 0.Narayan. 1976. Proteins specified by herpes simplex virus. J. Virol.

Membrane changes associated with assembly of visna 9:431-439.

virus.Virology 74:520-530. 26. Lowry, S. P., D. L. Bronson, and W. E. Rawls. 1971. 14. Duff,R., and F. Rapp. 1973. Oncogenic transformation Characterization of the abortive infection of chick em-of hamster embryo cells after exposure toinactivated bryo cellsbyherpesvirustype I. J.Gen. Virol. 2:47-51. herpessimplexvirustype1.J.Virol. 12:209-217. 27. Madden, D. L, K. E. Moats, W. T. London, E. B. 15. DuyckinckSmith, J., andE.de Harven. 1973.Herpes Matthew, and J. L. Sever. 1974. Mycoplasma moatsii, simplex virus and human cytomegalovirus replication anewspecies isolated from recently imported grivit inWI-38cells. J. Virol. 12:919-930. monkeys (Cercopithecus aethiops).Int. J.Syst. Bacte-16. Ejercito, P. M., E. D. Kieff, and B.Roizman. 1968. riol.24:459-464.

Characterization of herpes simplex virus strains differ- 28. Morgan, C., H. M. Rose, M. Holden, and E. P. Jones. ing to their effects on social behavior of infected cells. 1959. Electron microscopicobservation on the devel-J.Gen.Virol. 2:357-364. opment of herpes simplex virus. J. Gen. Virol. 17. Fitzgerald,S.C., D. A. Fuccillo, F. Moder, and J. L 110:643-656.

Sever.1974.Utilization ofafurther miniaturized sero- 29. Nii, S., C. Morgan, and H. M. Rose. 1968. Electron logical microtechnique.Appl. Microbiol. 27:440-441. microscopy of herpes simplex virus. II. Sequence of 18.Fong, C. K. Y., andG.D.Hsiung. 1972. Development development. J. Virol. 2:517-536.

of anequineherpesvirus in two cellculture systems: 30. Nii, S., H. M. Rose, and K. C. Hsu. 1968. Electron light and electron microscopy. Infect. Immun. microscopy ofherpes simplexvirus. IV. Studies with 6:865-876. ferritin-conjugatedantibodies. J. Virol. 2:1172-1184. 19.Fong,C. K. Y., R. B. Tenser, G. D. Hsiung, and P. A. 31. Roiman, B.,and D.Furlong.1974.Thereplication of

Gross.1973.Ultrastructural studies of theevelopment herpesviruses, p. 22940. In H. Fraenkel-Conratand andrelease ofguineapig herpes-likevirus in cultured R. R. Wagner (ed.), Comprehensive virology, vol. 3. cells.Virology 52:486-477. PlenumPress, New York.

20. Fraser, C. E. O., L. V.Mendez,and T. Simeone. 1974. 32. Schwartz, J., and B.Roiznan. 1969. Similarities and

Specificitydifferentiation ofherpessimplex virus types differences in thedevelopmentoflaboratorystrainsand Iand IIby indirect immunofluorescence. J. Infect. Dis. freshlyisolated strains ofherpes simplex virus in HEP-30:63-66. 2cells: electronmicroscopy.J.Virol.4:879-889.

21. Fuccillo, D.A.,F.Moder, L. W. Catalano, Jr., M. M. 33. Sheffield,J.B.1973.Envelopeof mousemammary tumor Vincent, and J. LSever.1973.Herpesvirushominis virus studiedby freeze-etchingand freeze-fracture tech-types I and II:aspecificmicroindirecthemagglutination niques.J. Virol. 12:616-624.

test.Proc. Soc.Exp. Biol. Med. 133:735-739. 34. Tillack,T.W.,R. E.Scott,and V. T. Marchesi. 1972. 22. Gershon,A.,L.Cosio,and P. A. Brunell. 1973. Obser- Thestructure oferythrocytemembranes studied by vations on the growth ofvaricella-zostervirus in human freeze-etching.II.Localization of receptors for phyto-diploid cells. J. Gen. Virol. 18:21-31. hemagglutinationand influenza virustothe intramem-23. Haines, H., and R. J. Baerwald. 1976. Nuclear mem- branousparticles.J.Exp.Med.135:1209-1227.

branechangesinherpessimplexvirus-infected BHK-21 35.Zee, Y.C., and L Talens. 1972. Electronmicroscopic

cellsasseenby freeze-fracture. J. Virol. 17:1038-1042. studiesonthedevelopmentof infectious bovine rhino-24. Heine,J.W.,and B. Roizman.1973.Proteinsspecified tracheitis virus in bovine kidneycells. J. Gen.Virol.

by herpessimplexvirus. IX.Contiguity of host and viral 17:333-336.

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Figure

FiG. blasts.virus1. Growth curve showing the titers ofreleased for the Patton strain of HSV-I in human fibro- M{ultiplicity of infection = 10PFU/cell.
FIG.2.thefaceouterThismembranecircular FF replica of virions released in the extracellular space (asterisk) at 36 h p.i
TABLE 1. Proportion of virions with heterogeneityin their envelopes at different times after viral
FIG. 5.granulesviral Portion of the nucleoplasm of an infected cell that was cross-fractured Viral capsids appear as arranged in a circle, sometimes enclosing a group of central granules probably corresponding to the core (arrows)
+6

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