Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

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0022-538X/80/12-0816/13$02.00/0

Analysis

of

Herpes

Simplex

Virus

Nucleoprotein

Complexes

Extracted

from Infected Cells

PIER F. PIGNATTII* AND ENZO CASSAI2

Istituto diGenetica, Universitd diPavia,27100Pavia,'andIstituto diMicrobiologia, Universitadi

Ferrara,

44100

Ferrara,2

Italy

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with

[3H]thymidine and '4C-amino acids. Infected cells or nucleipreparedfromthem

wereextractedwithTriton X-100 andNaCl,utilizingamethod

recently

described,

and thelow-speed supernatant(extract) waspartially purified bysedimentation on sucrosegradients. Anucleoproteincomplexwhichsedimentedas awidepeak

around 200S was identified. The

nucleoprotein complex

contained viral DNA,

which bandedattheexpecteddensityinCsClisopycnic gradientsandwasintact

after measurements taken on electron microscopic

photographic

enlargements.

Theautoradiographic patternof

"C-labeled

proteinsafter

electrophoresis

showed

thatonlya few of thevirus-specificpolypeptideswerepresentin thenucleoprotein

complexes,inparticular, VP5, VP12,VP15.2, VP19, andVP24.Cellularhistones

were absent. The extractsand thenucleoprotein complexes werecentrifugedto

equilibriuminmetrizamidedensity gradientswithoutprefixation. Electron

micro-scopic direct visualization of thenucleoprotein complexesaftersucrose or

metri-zamidepurificationrevealed thattheproteinswere

preferentially

associatedwith

oneendofthe DNAmolecule andformed

large irregular

terminal

thickenings

or

capsid-like transparent shellsenclosingpolyglobularcores.Nonucleosomeswere

observed on herpes simplex virus nucleoprotein complexes. The same type of

complexwas detected after phosphonoacetic acid addition, and grossly altered

nucleocapsidswereformed.

Nucleoprotein complexes (NPC) of small

DNA tumor viruses showing chromatin-like

structureshave been isolated fromcellsinfected

with polyoma virus (4) and simian virus 40 (4,

10,22)orfrompapilloma (7)and BK(18)virions.

The isolation of soluble minichromosomes has

greatly accelerated structural and functional

studies of these viruses. As to medium-sized

DNA tumor viruses, the replicative

intermedi-ates of adenoviruses show a different type of

structure which is devoid ofnucleosomes (15).

For the class oflarge DNA tumor viruses, the

herpesviruses, no such soluble complexeshave

asyetbeendescribed.

Herpesvirions areschematicallycomposedof

a centrally located core surrounded by three

concentricstructures: aproteincapsid about 100

nmindiameter,thetegument, and theenvelope

(23). Electronmicroscopicobservation ofherpes

simplex virus (HSV)-purified nucleocapsids (8)

andofthin sections ofHSV-infected cells (8, 12,

19) has allowed the identification ofviral core

particles containing DNA which might be

spooled aroundacentral mass ofproteins.

Tolook for the presence of HSV NPCduring

productiveinfection,wehaveused apreparation

methodwedescribedrecently (21) which allows

the rapid extraction of HSV DNA from the

infected cells without contaminationwith

cellu-larchromatin. This paper reports the isolation

ofHSV NPC and theanalysis of theirstructure

and composition. A tentative interpretation of

the NPC role in the growth cycle of

herpesvi-ruses based on present data and on previous

results is

provided.

MATERIALS AND METHODS Cell culture and virus infection. HEp-2 cells weregrown in Eagle minimal essential medium sup-plemented with 10%fetal calfserum.When thecells approached confluency, theywereinfected withHSV type 1 (HSV-1), strain F (6),at amultiplicityof1to 5 PFU/cellfor 2 hinphosphate-bufferedsaline supple-mented with 1%inactivatedcalf serum. After aspira-tion of the viralinoculumandwashing,thecells were maintained inEagle minimal essential medium sup-plemented with 1% dialyzed fetal calfserum and la-beled for the times stated in each experiment with

['3H]thymidine (5 1Ci/ml). Labelingmedium with

"C-amino acids (0.7,ICi/ml) contained one-tenth ofthe normalaminoacid concentration. The cellmonolayers were washedtwice with ice-cold phosphate-buffered saline, scraped witharubberpoliceman,collectedby low-speed centrifugation, andfrozenat-80°C.

Preparation of NPC. Extractswere prepared as described previously(21)bythawinginfected cell sam-plesand mixing them with9volumes oflysing solution containing 10mMTris-hydrochloride (pH7.9)-0.25%

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HSV DNA-PROTEIN COMPLEXES 817

Triton X-100-0.2 M NaCI-l0mM EDTA. Infected-cell nuclei were prepared according to the technique of Burgoyne et al. (3), and then extracts were prepared as described above. The suspension was clarified by low-speed centrifugation, and the supernatant (ex-tract) waslayered on linear 20 to 40% (wt/vol) sucrose gradients with 0.6ml of a 77% (wt/vol) sucrose cushion which contained0.01MTris-hydrochloride (pH 7.9)-0.2 MNaCI-1 mM EDTA. The gradients were centri-fuged in anSW40rotor at35,000 rpm and 4°C for 30 min.Fractions werecollected from the bottom of the tube,and acid-insolubleradioactivity was determined bytheglass fiber filter procedure (2).

Cytoplasmic nucleocapsids were prepared with the detergent method ofGibson and Roizman (9) and then weresubjected to velocity sedimentation analysis as describedpreviously.

CsClequilibrium density gradients. The DNA samplesweredeproteinized by incubation in the pres-ence of 100 ug of proteinase K per ml-0.2% sodium dodecyl sulfate-l0 mM EDTA-10 mM Tris-hydro-chloride(pH 7.5) for2hat37°C. CsClwasthen added and thesolutionwasadjustedto adensity of 1.720 g/

cm3. The DNA solution was centrifuged in a 65 Ti rotor at 40,000 rpm and20°C for 62 h, and fractions of 0.16mlwere collected from the bottom of the tube. Acid-insoluble radioactivity was counted as above. Ethidium bromide fluorescence was scored under a UVlamp after spottingasample of each fraction on WhatmanGF/C filters and soaking them inasolution containing0.5,ugof ethidiumbromide per ml in Tris-hydrochloride (pH 7.5)-lmMEDTA.

Metrizamide density gradients. Metrizamide

(Nyegaard & Co., Oslo,Norway) solutions were pre-pared inabuffercontaining 10 mM Tris-hydrochloride (pH 7.5)-0.2 M NaCl-I mMEDTA, filtered onglass fiber paper(GF/A Whatman) filters,andadjustedto pH 7.5 with NaOH. Discontinuous gradients were preparedasdescribedpreviously(22) andcentrifuged in the65 Ti rotor at 40,000 rpm and4°C for 39 h. Fractions werecollected,anddensity wasdetermined accordingtotheformula p=3.350c-3.462(1),where cis the refractive index.

Acrylamide gel electrophoresis and

autora-diography. Virion polypeptides (VP) and

infected-cell polypeptides were prepared as described

previ-ously (11, 13). Nucleoprotein sampleswereprepared

by dilutingappropriate sucrosegradient fractions in 10mMTris-hydrochloride (pH 7.5) and bypelleting

the NPC in the65Tirotor at40,000 rpm and4°C for 3 h. The pellets were thencollected and treated as described above. Calfthymushistonespurchasedfrom

Sigma Chemical Co. (St. Louis, Mo.) were used as

electrophoreticstandards.Electrophoresis onsodium

dodecyl sulfate-containing polyacrylamide gel slabs

cross-linked withdiallyltartardiamideandgel analysis

wereperformedasdescribedpreviously(11).

Electron microscopy. NPC were prepared for electronmicroscopic observation withoutprior fixa-tion,using theproteinlessmethod of Dubochetetal. (5)asdescribed previously (4, 22).Theaqueous mi-crodroptechniqueofKleinschmidt (17)wasalso uti-lized for DNA visualization. The samples were ob-served withaPhilipselectronmicroscopeEM300at

60kV,andphotographsweretakenatmagnifications

rangingfromx14,700 to x36,100. Measurements were done on photographic enlargements: diameters were determined with a lenswith0.1-mm graduations, and DNA contour lengths were determined with a map ruler. Calibration was based on examination of cross-grating replicas (Polaron Equipment Ltd., Watford, England).

RESULTS

Analysis of the extracts. HEp-2 cells were

infected with HSV-1, grown in thepresence of

[3H]thymidine, collected, and extracted with

Triton X-100 and NaCl-containingbuffer at

var-ious times after infection. In the extracts only

onepeak of DNAwas present, and it banded at

1.726g/cm3inCsCl gradients (Fig. 1A), a density

correspondingtotheexpected HSV-1 (strain F) DNAvalue (16). The absence ofcellularDNA

in the extracts was confirmed by the lack of

ethidium bromidefluorescence in the

appropri-ate gradient fractions (Fig. 1A), and it is in

accord with previous results(21)obtained with

cells which had been labeled with

['4C]thymi-dine before viral infection. The extracts

con-tainedincreasingamountsof viralDNA atlater timesafter infection.

Preparation andpreliminary characteri-zation of NPC.

Samples

of the extracts were

layeredon 20 to40%sucrosegradientsand

cen-trifugedasdescribed above.Abroadpeak

sedi-menting

atabout 200S

appeared (Fig. 2A),

which could be shiftedtothe HSV DNA sedimentation value afterproteinase K deproteinization (21). NPC were absent in cells collected 3 and 6 h

postinfection, appeared at 9 h, and then were

present atall latertimes

tested, i.e.,

12, 18, 32,

and48hafter

infection,

when cell

lysis usually

occurred. Thesamesedimentation

velocity

was

observed for NPC obtained at different times after infection.

Figure

2B shows theresults ofan

extraction fromanuclear

preparation.

AnNPC with thesamesedimentation velocityasin Fig.

2A was

identified;

the

peak

was

broader,

and

relatively moreviral DNA (in fractions 33 and 34) and mature

nucleocapsids (on

the sucrose

cushion in fractions4 and

5)

werepresent. The DNA from the

sucrose-purified

NPC bandedin

CsCl

isopycnic

gradients

as a

single species

dis-playing viralDNA density (see

Fig.

1B). DNA

molecules about45

pm

long

were

predominantly

observed in the NPC peak after

deproteiniza-tion. Figure 3 shows one such

filament,

which

wasfoundtobe 46.2

,m long.

Proteins present in NPC. To

analyze

the

protein component of the

complex,

infected

HEp-2 cellswere labeled with

14C-amino

acids

and then

collected;

theextracts were

subjected

to

velocity sedimentation

as described above.

Figure 4 shows that, whereas most

14C-labeled

VOL. 36,1980

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818 PIGNATTI AND CASSAI

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FRACTION

NUMBER.

FIG. 1. Equilibriumdens.tycentrifugationprofilesofthe DNApresentintheextract(A)and in the NPC (B). The cellswerelabeled from5to 18hafter infectionwith[3H]thymidine, andthenextraction, sucrose gradientpurification,sampledeproteinization, andCsClcentrifugationwereperformedasdescribed in the text.(A) Triton-NaClextrac&; (B)pooled fractions15to22ofthegradientshowninFig. 2A,dialyzed against

10mMTris-hydrochloride (pH 7.5)-10mM EDTAfor16hat4'C.Arrows indicate observedpeak density

(1.726 and 1.722g/cm3) or expected cell DNA density (1.703and 1.702g/cm3), as determined inparallel

gradients.

polypeptidespresentintheextractweresoluble

and did not penetrateint-o the

gradient,

about

1%of the labeledprotein

pool

waspresentinthe

NPC

peak,

which coincidedwith the [3H]DNA

peakshown inFig. 4D.All experimentinwhich

14C-labeled

amino acids werealreadypresent in

the medium24hbefore viral infection

(Fig.

4B)

gaveresults similar tothose shown in

Fig.

4A,

and in

particular,

noincreaseintheNPC

radio-activity peak was observed. The

analysis

ofa

control uninfected cell extract prepared at the

sametime after"4C-aminoacid

labeling

for48h

(Fig.4C) showed thatno cellularproteins

sedi-mented at around200S in the absence of viral

infection, but all remained at the top of the gradient.

To determine the protein species associated

with viral DNA in the NPC, the appropriate

sucrosegradientfractionswerediluted, pelleted,

electrophoresed,andautoradiographed.The

au-toradiographic pattern of

"C-labeled

proteins

presentin theNPCis shown inFig. 5.Itcanbe

noted that all of theproteinspresentinthe NPC

(Fig. 5, lane B) isoelectrophoresed with some

corresponding infected-cell and virion

polypep-tides. From the analysis of the electrophoretic

migration rates in this and other gels, the follow-ing conclusions were reached: VP5, -12, -15.2,

-19,and-24werealways present as clear bands

in theNPC. In addition, some

small-molecular-weight polypeptides, perhaps degradation

prod-ucts, migrated with the electrophoretic front.

Sometimes traces ofVP7, VP8.5, VP13, VP14,

and VP16werealso detected. It should be noted

thatmanyvirion polypeptides were always

ab-sent from the complex, in particular, some of

those represented most often in the viral

parti-cles, suchasVP8, VP17, VP18, and VP22. The

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A

I II

S I I

4 5 40 45 to Zs 30 35

B

4 5 40 15 20 25 s0 35

FRArTION N UM

FIG. 2. Sedimentationvelocity profiles of HSV-infectedcellular(A)ornuclear(B)extracts. Infectedcells

werelabeledfrom5 to 18 hafter infectionwith[3H]thymidine, collected,and dividedinto twosamples.One

samplewasextracteddirectly (A),whereasfromthe othersamplenucleiwerefirstisolated andthen thesame

extractionprocedurewasapplied (B).The twosamplesweredepositedontosucrosegradientsandcentrifuged asdescribed in thetext.Directionofsedimentation isfrom righttoleft.Thearrowindicates the sedimentation position ofanHSV-1(strain F)DNA marker.

NPCwas thusparticularly rich inthree capsid

proteins (VP5, VP19,andVP24),whereas itwas

devoid ofothers, e.g.,VP22.

Slightlymodifiedgelsin whichtheacrylamide

concentration was raised from 8.5 to 15% were

utilized for a better separation of cellular his-tones. One such electrophoretic analysis is shown in Fig. 6. Histones (Fig. 6, D) were not detected in HSV-1 NPC (Fig. 6, F)orinvirions

(Fig. 6, E),buttheywerepresentinthe

infected-cellpolypeptides (Fig. 6, G).

A searchwasmade forenzymesand cofactors retained in the NPC by investigating

endoge-nous DNA and RNApolymerizingactivities in

the sucrose gradient fractions. Cellulara, fi, y,

and HSV DNApolymerases were allpresent in

the extracts (20),whereas noendogenous HSV

DNA synthetic activity was detected in the

NPC. Endogenous RNA polymerase activity

wasdeterminedaccordingtoWilhelmetal.(25):

no [3H]uridine monophosphate was

incorpo-rated bythe viral NPC fractions. Extraction of the infected cells with 0.4 MNaClordialysisof

pooledfractionsdid notchangethe results.

Structure of the NPC. Samples obtained from thesucrosegradientfractions

correspond- I-v

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6'

0

b Q

42, J6 is,~ .4, .&,

411~~~~~~~~~~~~~~~~~~~

:,¶,..

*

4

.~~ *%.ILA *0~

*2% J:..7.

FIG. 3. Electronmicrograph ofan intactHSVDNAmoleculepresentintheNPCpreparation. Kleinschmidt (17)procedure. Bar,0.5 Wn.

ingtotheNPCpeakwereutilized withoutprior wereinterpreted to be slightly expanded,

capsid-fixation for electron

microscopic observation,

us- like shellsenclosingviral cores. The core

glob-ingthetechnique ofDubochetetal. (5). Afew ules weresometimeslinearly arranged (see Fig.

examplesofthestructureswhichweredetected 7A and B), but more often were in groups of

areshown inFig.7AtoG. Electron-denseirreg- three at an angle to each other and with a

ularglobulararrayswerepresent insideroughly common central contact (see Fig. 7C to G).

hexagonal electron-translucent capsules, with About 90%of the particles had DNA emerging

diametersrangingfrom 110 to 120 nm (Fig. 7B from them with noproteins associated (Fig. 7A

toF).Exceptionally, amuchlarger particle(ca. to E).Often two distinct DNA filaments clearly

150nm) wasobserved (Fig. 7G). As acompari- appeared to enter and exit from the described

son, viral nucleocapsids observed under the structure (Fig. 7A, D, and E). Exact

measure-sameconditions showed diameters of about100 ments of DNAcontour length were quite

diffi-nm(Fig. 7H andI).Thenucleoprotein particles cult with thesepreparations: the filament shown

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PIGNATTI AND CASSAI

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VOL. 36, 1980

in Fig. 7A was about 28 jim long; other values

obtainedwerein the rangeof22to 29jim,except in one case in which a 43-,im-long filament was observed. In about 10% of the cases, no DNA

filament appeared to originate from the

parti-cles,asfromthoseshown inFig. 7F and G.

Extracts and sucrose gradient-purified

com-plexesweresubjected to isopycnic centrifugation inmetrizamide densitygradients, a method that

allows nucleoprotein density determinations

IX 0J6 WI 04 S OLa IL U0 W

2I

2-i

III a A

3a

I I o u .5 3 .4 FRACTION NUMAEA

FIG. 4. Sedimentationvelocityprofiles of

HSV-in-fectedcell extracts.(A) Cells labeledfrom5to 18 h after infectionwith

"4C-amino

acids. (B) Cells

incu-batedfor24 hbefore infectionandfrom5to 18 hafter infectionwith14C-aminoacids.(C)Controluninfected cells labeledinparallel for48 hwith

"4C-amino

acids. (D) Cells grown in the presence of[3H]thymidine from5to18 hafter infection.Allcellswerecollected

atthesametime, extracted, andcentrifuged as

de-scribedin thetext.Directionofsedimentation isfrom righttoleft.

A R0

VP

1-2

5 m '

8 8.57 -12 13

is4

A 15.2 16 AM 17 18 19 22 23 24 A IJ t. ICP ft~ 2 faw.. 4a-c 5 7 8 10 12 17 20 24 25 29 32 33 36 39 40 41 _ 43 44

FIG. 5. Autoradiogram oftheproteinspresentin

the NPCafter electrophoresisonpolyacrylamide gels.

(A) HSV-1 (strain F) virion polypeptides (VP) ob-tained from 4C-amino acid-labeled virions, num-beredaccording toHeineetal. (11). (B)NPC

poly-peptides obtainedfrom fractions 16and 17 ofthe

sucrosegradientshown inFig.4A. (C)Infected-cell "4C-labeledpolypeptides (ICP) obtained 18 h after

infection,numberedaccordingtoHoness and

Roiz-man(13).Molecularweightsofselectedpolypeptides

aregivenas areference (13): VP5= 155,000; VP12

= 87,000; VP19 = 53,000; VP24 = 25,000. Sample

preparation, electrophoresis, and autoradiography

wereperformedasdescribed in thetext.

without prior fixation of the complexes. The

extract (Fig. 8A) showedthe presence of a main

nucleoprotein peak atabout 1.15

g/cm3,

with a

heavier shoulderat 1.20

g/cm3

and alighter peak

at 1.12

g/cm3,

which probably corresponds to

free DNA (1). The sucrose-purified NPC (Fig.

8B) showed a single peak at about 1.15

g/cm3.

Asthebuoyant density in metrizamide depends

upon the ratio ofproteins to nucleic acids in the

complexes,itwascalculated (according to Birnie

etal.[1]) thatthe HSV NPC contained slightly

fewerproteins than DNA by weight. For

com-parison, it can berecalled that purified virions have a protein-to-DNA weight ratio of about

10.7 (11). Samples from the metrizamide

gra-wp

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822

PIGNATTI AND CASSAI

D

E

F

G

(VP)

12 15 19c 24

FIG. 6. Electrophoretic analysis of the polypep-tides present in the NPC after electrophoresis on modified gels andstaining. (D)Histones. Black ar-rows indicate histone Hl and histones H2A, H2B, H3, H4, respectively. (E, F,andG)SameasFig. 5A, B, and C, respectively. Clear arrow indicates the position ofhistone bandsin(G). Sample preparation andelectrophoresis wereperformed asdescribed in thetext.

dient fractions were directly prepared for

elec-tronmicroscopyasdescribed.Someexamplesof

the observed structures are shown in Fig. 9.

Proteinswere locatedmostly atone endofthe

DNAfilament, asshownintheoverallviews of

Fig. 9A andB.Figures9C to Fareenlargements

ofprotein-associatedDNAterminifrom

metri-zamidefractionsshowing large (upto about 500

nm in length) masses with globular

substruc-tures.Thebackgrounddebriswasinterpretedto

be smallribonucleoprotein particleswhich

sed-iment at about the same density as the HSV NPC in metrizamide gradients (14).The DNA

filamentemergingfrom such nucleoprotein

ag-gregatesor"heads" almostinvariablyshoweda smaller(Fig. 9A) orlarger (Fig. 9B) loop,

some-timesfoldingbackonitself almostentirely,asin

Fig. 9B. Thefilament shown in Fig.9A was27

pmlong; thatin Fig.9Bwas25pmlong. Other

lengthmeasuresfell between 25 and 30,m.

Phosphonoaceticacid inhibition ofvirion

biosynthesis. To searchfor different kinds of

NPCduringvirusformation,infectedcells were

growninthepresenceofphosphonoacetic acid,

an inhibitor of HSV DNA

synthesis

and virus assembly. In an experiment in which 100,g of phosphonoacetic acid perml was given at 6 h and the cells were then collected 24 h after infection, the percentage of

[3H]thymidine

in-corporation into acid-precipitable DNA was

about7.5,whereas itwas28% in controlcellsnot

treated with the drug. When the extracts were

analyzedonslightly modified velocity sedimen-tation gradients (Fig. 10), very similar nucleo-protein peakswereevident both in thepresence

(Fig. 10A) and in the absence (Fig. 10C) of phosphonoacetic acid. From thesamecells

viri-ons wereprepared accordingtoGibson and

Roiz-man(9)andsubjectedtovelocity sedimentation analysis (Fig. 10B and D). A newheavier sedi-mentingpeak of NPC appeared in the phospho-noacetic acid-treated cells (Fig. 10B), whereas nucleocapsids accumulatedonthesucrose cush-ion inthe untreated control (Fig. 10D).

Electronmicroscopic observation of the peak fraction of Fig. 10B revealed the presence of

aberrant nucleocapsids,asshownin Fig. 11. Two

differentgroupscouldbeidentified: the first less well represented group consisted of particles

with diametersof about110 to 120nmexhibiting

structural alterations, the most common one

being the bendingoutofoneside of their

shell,

as shown inFig. lld and f. The second,larger

groupincluded particles whichwere about half

aslarge (widths ranging from50 to 75nm) and

altered: sometimes the above-described

protru-sionswereobserved, asinFig. llband e;

some-times more uniform variants were seen, as in

Fig. lla, c, and g; and sometimes miniature

capsids about 50 nm insize and with irregular

sidelengthswereseen, as inFig.llhand i.

DISCUSSION

HSV NPC have been isolated from infected cells andcharacterizedby using thesame

meth-ods utilizedpreviously (4, 22) forthe extraction

andanalysis ofpolyoma virus and simian virus 40minichromosomes from infected cellsand

nu-clei. Viral complexes devoid ofchromatin

con-tamination wereextracted similarly, but

differ-ences in NPC composition and structure were

observed. Onlysomevirus-codedproteins were

associatedwithHSV DNA, whereascellular

his-toneswere bound to polyoma virus and

simian

virus 40 genomes. Moreover, electron

micro-scopic

visualization

showed that the HSV

poly-peptides were located primarily at one end of

the DNA molecule, whereas on the contrary,

nucleosomes were distributed uniformly in the

small viralminichromosomes mentioned above.

HSVDNAthusseems to be an exception to the

eucaryotic "nucleosome rule,"possibly together

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824

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FRACTION NUMBER.

FIG. 8. Metrizamidedensity gradientanalysis ofacellextract(A)andNPCfrom pooledsucrosegradient

fractions (B). Infected cells werelabeled with[3H]thymidine from 6to 19hafter infection, collected, and extractedasdescribed in thetext.Discontinuousmetrizamidegradientswerepreparedandrun asdetailed in thetext.

with another class of DNA tumor

viruses,

the

adenoviruses (15).

Concerningthe virusgrowth cycle,wepropose

thefollowinginterpretationofourdata.(i) Upon

HSV-1 infection of HEp-2 cells in culture, a

DNAreplicationcomplexisformedfirst,similar

tothatwehave

prepared

with thesamemethods

from HSV-1-infected HEp-2 cells (20) which

contains viral DNApolymerase and other

fac-torssufficient for in vitro continuation of DNA

synthesis. (ii) Structural components are then

synthesized, and the DNA ispacked intoNPC

similar in appearance andprotein composition

to those described here. As the DNA "tails"

emerging from the nucleoprotein heads were

usually shorter than expected full length (24),

whereas afterdeproteinization45-,um-long

mol-eculeswere

observed,

wesuggestthatsomeviral DNAmightbecompactedinthe heads. Nucleo-capsid proteins VP5, VP15.2, VP19, and VP24

could begood candidates for the role of starting

viral DNA encapsidation in vivo. As an

alter-nativeinterpretation,ifcapsidswerepreformed,

there mightbe smallamountsofother proteins

which "compact" the DNA into the capsid. We

would not detect such proteins if they were

present in only a few copies per capsid. The

NPC described here, which aredevoid of DNA

replicatingandtranscribingactivity, might

rep-resentbotha naturalintermediate in virion

as-sembly and a somehow more stable form in

particle disassembly, obtained by a controlled

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826 PIGNATTI AND CASSAI

rRACTION NUMBER

FIG. 10. Sedimentationanalysisoftheeffectsofphosphonoaceticacid additiontoHSV-infectedcells.Cells weregrown in the presence (A and B) and in the absence (C and D) of phosphonoacetic acid.

[3H]-thymidinewas added 6 hafter infection. Cellswerecollected24hafter infection.NPC(A andC) and virion (B and D)preparationprocedures were then applied. The samples were layered onto 20 to 50%sucrose gradientson77% cushions andcentrifugedat30,000 rpm and4°Cfor25min.Otherproceduresaredescribed

inthetext.Directionof sedimentation isfrom righttoleft.

disruption process. It does not seemlikely that

theNPC are artifacts of thepreparation

proce-dures,astheyareabsentfrom uninfected cells,

theirquantityincreases withtime after infection,

they are present also in isolated nuclei, they

appear inreduced amounts when virion

biosyn-thesis is reducedbyphosphonoacetate,andthey

can beobserved afterappropriate treatment of

isolated cytoplasmic virions. Moreover, as can

be inferred from other examples, very similar preparation procedures have yielded

informa-tiononpolyomavirus andsimian virus40NPC

inside the cell which have been confirmedbya

variety of different methods.(iii)TheNPC could

finallyassemble intonucleocapsids,astepwhich

canbe inhibitedbyphosphonoacetic acid,which

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University for theuseof their electronmicroscope,andCesare

Mussi fortechnical assistance.

[image:12.496.34.225.67.286.2]

f

FIG. 11. Electron micrographs of

paredfromcellsinfected withHSVwc

presence ofphosphonoacetate.

Samp

from fraction19of thesucrosegradiei

JOB and prepared by the techniqueof

(5).Bar,50nm.

allowsthebiosynthetic processto

alittlebeyondstepii while variant

areformed, probably for the lack

essarystructural polypeptides. TI tionconstants of the complexespr

1,ii,andiiishowaprogressivemcr

value of naked viral DNA to th nucleocapsids. This could be ex

greatercompactnessobtained by t addition of the appropriate vira DNA. We should finally mention lication complex (step i) activit3 already detected incellscollected

tion, reaching itsmaximumvalue.

decliningthereafter to lowerlevel tionofNPC, onthe contrary

(ste]

a different time course: they firsi

cells collected 9 h postinfection creased in quantity upto 18h. Th of HSV NPC appearance in the

thus closely parallels that of viric

(23).

ACKNOWLEDGMENTS This work was supported by Consiglio

Ricerche (CNR) contribution CT 78.01424.' gettoFinalizzato Virusgrant79.00379.84,an

1225.

We thank Bernard Roizman for helpful constructive criticism, the CNR Centro l'Istochimicaand theIstitutodi AnatomiaCc

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J. VIROL.