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
20014Received 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 changeswerecompared 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 probablyrepresentvirus-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 facescovered 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 ofthefreeze-fracture techniquetoherpessimplexvirus type1-infectedcells revealed
striking
structural differences between viral and uninfected cell membranes. These differencesareprobably
relatedtoinsertion andclustering
ofvirus-coded proteins in thehydrophobic
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 haveprovided
evidence the inner nuclear membrane or sometimes at thatthe virus budsthrough newly
synthesized
cytoplasmic membranes. The enveloped virion regions ofthenuclear membranecontaining
vi-containsatleast24
specific
proteins,suggesting
mus-specific proteins
andglycoproteins
(5). Inthat
assembly
is acomplex
process (31). Onceaddition,
newantigens
thatareidenticaltothosethe capsid has been assembled in the nucleus, oftheviral
envelopes
have been detectedonthesome 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, theare seencloseto
protruding regions
ofthe inner freeze-fracture(FF) technique
wasapplied
tonuclear membrane which has a thin
layer
of HSV-1-infected cells. Thistechnique splits
cel-electron-dense material under its inner leaflet lular and viral membranesbetweenthetwo leaf-and will form theenvelope
of the virus. The lets and revealsintramembraneparticles (IMP)
enveloped
virus travels within a vacuole fromthought
tocorrespond
toproteins
and notde-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 alterationsweredetectedspikes onitssurface. Insome cell systems, the in several viral structures and
thought
to beplasmalemma 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 wereequili-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
ofminat37°C to allow viral penetration.
Vion
owth
ce.
U s asynchrof
The nonabsorbed viruses wereremoved, and cells infectionof 10PFU/cell, all cells were synchro-wererefed with minimumessential medium containing
nously
infectedwith thePattonstrainofHSV-1antibiotics 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 hImmunofluorescent 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 thepro-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 membranechangesoccuring
more, Md.). during
HSV-1anfcll
wereangesucces-Electron microscopy. Viral maturation of two
dursig
and infection were analyzedsucces-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 viralenvelope
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 same2_
/structural
featuresallovertheirenvelope.
In theX-
/FF
replicas,IMPwereabsent on one pole of the103/
innerhalf of theenvelope
ofsomeviruses(Fig.
2and
3d,
andsee E inFig.
16).
Thedensity
ofIMP on these inner leaflets was 2,329 ± 870
102
IMP/,um2
whenareaswithout IMPwereconsid-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 membrane0 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 FFFiG.
1.Growth
curveshowing
thetiters
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 virionenvelope
might be relatedtodeg-at 12hp.i. with the VR3 strain.Intheplatinum radatory changes relatedto aging of the virus.
replicas
obtained afterfreeze-fracturing
infected Viralassembly.
In thinsections,
viralcap-cellsat 12 to 48hp.i.,
hemispherical
membranes sidswerefirstseeninthenucleoplasm
at8hp.i.
withmeandiameters of 164±12.7nm (standard with each viral strain used.
They
were eitherdeviation)
were observed in the extracellular scatteredoringroups, sometimesforming
crys-space(Fig. 2).
Theseconcaveandconvex mem- talline arrays(27, 28, 31).
Thesecapsids
werebraneswere
identified, respectively,
astheouterreadily
identifiedin theFFreplicas
of cells in-and innerleaflets of free viruses because of their fected for8hor more(Fig.
5) (23).Acluster of size andshape
aswell astheir absence around granulesfrequently
present inthecenterofthese uninfected control cells.Histograms
of the di-capsids
probably
corresponds
to the viralcore. ameters ofthesehemispherical
structures and Protrusions ofthe inner nuclear membrane those ofvirionsobservedinthin sections showed overaviralcapsid
wereobservedinthinsectionsasimilar distribution.
However,
the diameters of at 12 and 16 hp.i.
in cells infected with thevirions that had been embedded in
Epon
were Patton strain and at 16 to 48 hp.i.
incells
consistently
smaller than those that had been infected with the VR3 strain. In cells freeze-frozen inglycerol
and fractured(Fig. 4).
This fracturedatsimilar timesp.i.,
scatteredprotru-difference in sizewas
probably
duetotheshrink- sions with a well defined circular base were ageof the virusduring
thedehydration
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 ±37.2nrm
(Fig. 6, and see A inFig. 16). These viral envelope in the FFreplicas
was the in- protrusions were interpreted as viral buddingcreased 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, iton November 10, 2019 by guest
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-e
r.
.,
.4
N
v7
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]VOL. 26, 1978
439
50
EJThin
Sections8).
Much later in infection(48
hp.i.),
groupsof virionswereoccasionally
seenin theperinuclear
F. F. Replicascisternae,
although nuclear budding sites were40 rarely detected after 16 h p.i. (Fig. 9). Their
membrane was
strikingly
different from theearly
virions,
sincethey
had much more IMP per square micrometer as did theextracellular
30 >virion.Thissuggests thatsomevirionscanstay
z
z X fora
prolonged period
oftime
in theperinuclear
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 cell120 140 160 180 200 surface
(Fig. 8) (28).
The inner leaflet ofvirionsDIAMETER OF VIRAL ENVELOPES (nm) lying inside vacuoles was rarely uncovered by
thefracture processsothatnoquantitative
ap-FIG.4Hsgs
o ftiats
ofviral
evpreciation
of the IMPdensity
could be obtainedoeplicsobservedain)tinl
sections(oblique lines)nd
FF
for theextracellular virions. The inner leaflet of replicas (in black). Only virions which showed acapsidin 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- theperinuclear
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
IMPsimilar
insize
and and shape to pinocytotic vesicles (Fig.10a,
and density to those of the adjacent nuclear mem- see B3 in Fig. 16). These werenormally
scatteredbrane.
all over thecell
surface in uninfectedcells.
InChanges 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.).
Roundareaslacking
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 toclose to
budding
sites(Fig. 6) (23).
Inaddition,
the plasma membraneofthe limiting membraneclusters of IMPondiscrete
protrusions
oftheP ofthe vacuolecontaining the virions.face of the innernuclear membrane were seen Unenveloped capsids lying in the cytoplasm
occasionally
(Fig. 7).
Theseprotrusions
didnot and cytoplasmic envelopment were observed havethe size andshape
of the muchmorefre- only late in infection(36 and 48 hp.i.)
with both quentbudding
sites andmight
be related to strains of HSV-1. They were, however, morediscreteareasof
thickening
of the nuclearmem-braneseenin thin sections
(29).
TABLE 1. Proportion ofvirions withheterogeneityIndividual
virionsreleased in theperinuclear
in theirenvelopes
atdifferent
timesafter
viralspacewereobservedatthesame timethat the infection
nuclear
budding
sites were identified in cells Virions (%) showinginfected with the Patton strain(12and 16 hp.i.). Type of virions heterogeneityat: In the
replicas,
the inner leaflet of the viralenvelope was characterized
by
apopulation
of 12 hp.i.
48 hp.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)on November 10, 2019 by guest
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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 higheron November 10, 2019 by guest
http://jvi.asm.org/
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(ontheright)
arein closecontactwithdepressionsoftheplasmamembrane in thin section(a)and FF(b). Vesicles,which areformedby plasmamembrane invagination, appearedtobeclustered aroundthesedepressions. This is moreclearlyseenwhenthe Efaceofsuchplasmamembranehas beenlargelyuncoveredduring
thefracture
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
and48hp.i.).
Fusedmem-VR3 strain. The virus was seen
budding
frombranes,
oftenarranged
inaconcentricway,werethe
cytoplasm
into vacuoles which often con- observed in thecytoplasm
ofinfected cells and tainedcomplete
virions(Fig.
11inset).
IntheFF consisted ofpairs
ofunit membranes whicharereplicas, elevations of various altitudes
protrud-
probably
extensions of the nuclear membrane ing into the vacuolar lumenwere identified as(Fig. 12a) (6,
29,30).
Pairs ofmembraneswerebudding sitesonthe vacuolarPface
(Fig. 11).
Inseparated
by
anelectron-dense materialsimilarcontrast to the
budding
sites at the nuclear to the one observed atthe inner leaflet of the membrane, thePface of theseprotrusions
was viralenvelope
(Fig. 13a).
These concentric fused covered withahigher
density
ofIMPthan the membranes werereadily
identifiedin FFrepli-adjacent
vacuole membrane and resembled the cas.The fracture faces oftheleafletsapposed
toinnerleaflet of extracellular virions
(Fig.
11,and the electron-dense material were covered withseeC in
Fig. 16).
numerousandclosely packed
IMP ofadensity
Membrane
changes
not associated with similartothat of the innerleaflet ofextracellular viralmaturation. Membranechanges
notas- virions(3,078
± 783IMP/,um2) (Fig.
12b and sociated with viral maturationwereallobserved13b,
and see F inFig. 16).
Leaflets in contactmagnification,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
thebudding
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.on November 10, 2019 by guest
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[image:8.509.77.468.68.604.2]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
ofparticles
similartothatof the E branesurrounding
theseclusters, however,
did face of the nuclear membrane. not appear to differ from that of control cellTheP 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 couldcorrespond
tothe mod-strainspecific.
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 theFFtechnique
G in Fig. 16). These ridgeswerealmostdevoid and
compared
with data obtained from thinofIMP, 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:10.509.73.458.64.538.2]~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
W-INt,FIG.~~~~~~
14.
and 15.
Coprsnbtenpamlma
ffbolssifce
o
8hwt
hatnsri
7~,%
k- 4~~~~~~'.. 7
(Fig.
1s
fewer
IMPthn.hesurondngP
ac.heruracsowsfieilmetspaaleltothmjoaisofth :~~~~'~~~Ab~~~~A~~~~&,2 , -~~~~~~J:. oFaiG.s
14ze
and15sCmarisocn bedtwecen
plsatlemmas.hyaemd
fbolssiectelore48
ofhake
wIth.
thePatontraFin.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 aton November 10, 2019 by guest
http://jvi.asm.org/
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 anchoredviral 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
on November 10, 2019 by guest
http://jvi.asm.org/
[image:11.509.93.405.61.362.2]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 tobetterlayer 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, havebeen 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-
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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
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Thedefinite structural cells. Eur. J.Cancer9:263-272.characteristics
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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.Synthesisofmembranes. It is
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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
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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.
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studiedt (VRt3)
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