Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

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0022-538X/80/10-0353/08$02.00/0

Intracellular

Forms

of

Simian Virus 40

Nucleoprotein

Complexes

III. Study

of Histone

Modifications

M. COCA-PRADOS, G. VIDALI,'AND M.-T. HSU* TheRockefellerUniversity, New York, New York10021

Themodification patterns of histones present in variousfonrns of intracellular

simian virus 40 nucleoprotein complexes were analyzed by acetic

acid-urea-polyacrylamide gelelectrophoresis. The results showed thatdifferent viral nu-cleoproteincomplexes contain different histonepatterns. Simian virus 40

chro-matin, which contains the activities for the synthesis of viral RNA and DNA,

exhibits a histone modification pattern similar to that ofthe host chromatin.

However, virion assembly internediates and mature virions contain highly mod-ified histones. Pulse-chase experiments with

[3H]lysine

showed that the newly

incorporated histones in the virionassembly

internediates

were already

highly

modified. The majority of in vivo acetylation activity of histones occurred on the

70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate.

These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into

thematurevirion: (i) modification of histones, (ii) accumulation of capsid protein

around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion

assembly isdiscussed.

Insimianvirus 40 (SV40)andpolyoma virus,

viral DNAsareassociated with histones derived

from the host cells (6). The histones in the

mature virion, however, show both qualitative

andquantitative differences from thehistonesin

thehost chromatin. Besides the absence ofHi

histones,the histonesin mature SV40 and

pol-yoma virus were found to be acetylated to a

much higher degree than the cellular histones

(2-4, 9, 11, 17). Recently, the histones in the

intracellular viral nucleoprotein complexes (NPC)werealsofoundtobehighlymodified (2, 3).However,theviral NPCused in these studies

were obtained byextraction from the infected

cellswith Tritonbuffer,which wehave shown

to causedisruptionofmaturevirions and virion

assembly

intermediates.Since virions and virion

assemblyintermediates accumulate late in infec-tion,the 70S NPC studied by previous

investi-gatorsare probably

composed

ofboth the

gen-uine70S viral chromatin andthe disrupted

vir-ions. Thus, it is rather unclear whether the

highly modified histone patterns observedin the

70S NPC represent the true70S viral

chroma-tins.

In our earlier report we developed a

gentle

extraction method for

isolating

intact

intracel-lular formsof viral NPC(7).Three formsof viral

NPC could be obtained by this extraction

method:(i)maturevirus(V), (ii)virion

assembly

intermediate (NPII), and (iii) the 70S viral chro-matin (NPI) which is the precursor to NPII complex and contains the major activities for synthesizing viral DNA and RNA. The purpose

ofthepresentcommunication was to

reinvesti-gate the histone modification patterns in the

various forms of intracellular SV40 NPC

ex-tracted by the gentle procedure. The results

showed that the histones in mature virions and

virionassembly intermediates (NPII) were

mod-ifiedto amuchhigherextentthanthose ofSV40

and cellular chromatins. Furthermore, we

per-formedpulse-labeling experiments and showed

that extensive modification of histones in virion assemblyintermediatesoccurred before the

ac-cumulation of capsid proteins around SV40

chromatin.

MATERIALS AND METHODS

Cells and growth of SV40 virus. SV40 virus strain SVS was grown on CV-1 cells as described

previously(7).

Radioactive pulse-labeling of SV40-infected cells.Allpulse-labelingexperimentswereperformed

in a 370C room. Forpulse-labeling with [3H]lysine,

cells were first incubated for 10 min in lysine-free medium(GIBCOLaboratories)supplementedwith2%

dialyzedfetal calfserum. Themediumwasthen re-placed by a medium containing 200 to 400 !LCi of

[3H]lysine (60to80 Ci/mmol;Amersham Corp.)per ml for the period desired. At the end of the pulse 353

on November 10, 2019 by guest

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354 COCA-PRADOS, VIDALI, AND HSU

period, thepetri dishwasimmediatelyplacedonice,

and theradioactive mediumwasreplaced byice-cold Tris-buffered saline. For chase experiments with

ly-sine, theradioactive mediumwaswithdrawn,and the cells were washedoncewith10mlof medium contain-ing15mgofunlabeledlysine-HClpermland 2% fetal calf serum and overlaid with thesamemedium for the chaseperioddesired.

Cellswerepulse-labeledwith 5 mCi of[3H]acetate

(2to 5Ci/mmol;AmershamCorp.)perml for 10or30 min.Procedures forterminating pulse-labeling follow

thosedescribed for[3H]lysinepulse-labeling. Extraction ofintracellular SV40 NPC. SV40 NPCwereextractedfrom the nuclei ofSV40-infected cells,asdescribedpreviously(7).Briefly,infected cells werewashed withTris-bufferedsaline andlysedwith 0.5%Nonidet P-40. The nucleiweresuspendedinTD buffer (25 mM Tris-hydrochloride, pH 7.4, 0.136 M NaCl,7mMKC1,0.7mMNa2HPO4)andhomogenized

in a tight-fitting Dounce homogenizerfor about 30 strokes. Cell nuclei were pelleted by centrifugation

(1,000xg),and theextract waslayeredon a5 to40%

sucrosegradient containing10mMTris,pH7.4-0.5%

phenylmethylsulfonyl fluoride and spun in anSW40

rotorat37,000 rpmfor70min.

Gelelectrophoresisanalysisof histone. Discon-tinuous sodiumdodecylsulfate-gelelectrophoresiswas

performed bythemethod of Laemmli(10). Polyacryl-amidegels,12%(13by22cmby1.5mm),wereusedto

analyze histones present inSV40NPC. Forstudying

the histonemodificationpattern, the acid-urea-poly-acrylamide gel electrophoresissystemasmodifiedby

Shmatchenko andVarshavsky wasused (15). SV40 NPCweredissolved in9Murea-3%

mercaptoethanol-0.9Maceticacid-0.5%cetyltrimethylammonium bro-mideandelectrophoresedina15%polyacrylanidegel

(13by33 cmby1.5mm)at 17V for34hat4°C.Gels

werefixed in 50%trichloroacetic acidovernightat4°C

and stained with0.1%Coomassieblue in50% trichlo-roacetic acid. Destaining was achieved by repeated washings withasolutioncontaining5%methanol and 7% aceticacid. Gelsweresplicedinto 1-mmslices for radioactivecounting.

RESULTS

Electrophoretic patterns of histones in SV40 NPC. SV40 NPC were isolated from

SV40-infectedCV-1 cells at48hpostinfectionas

described previously(7). Theextractwasfurther

fractionated in a 5 to 40%sucrose gradient into three subspecies of viral NPC: (i) SV40 chro-matin (70S), (ii) replication intermediates (1OOS), and (iii) virion andvirion assembly

in-termediates(150 to210S). The histones present

in these various fractions of intracellular NPC

were analyzed by acetic

acid-urea-polyacryl-amide gel electrophoresis as described by

Pan-yimnandChalkley(12). This gel system is

partic-ularly useful in analyzing histone modification

patterns in which modified species migrate as

slower bands (8, 12). Such analysis reveals a

rather distinctive modification pattem of

his-tonespresentinSV40chromatinand in mature

virus purifiedfromCsClgradients (Fig. 1Band

C). The histonespresentinSV40chromatinare

lessmodified and resemble cellularhistone

pro-file(Fig. 1A and B), whereaspurifiedviruslacks

0 0

4-0_

I..

x

a-0

A H20

H2b

It

ItI

-1

H 1 i

1~~~I

11

a

4Ho

SHE

I

II II

H2

liii

1 II"

I,II 1

I

lIl

I

1 1'

B III 1l1i1i

Il It I

6~ ~~i II

3-~

~

I t li

2

0I

tl

O 10 20 30 40 50 60 7O

[image:2.499.268.439.145.596.2]

Fraction number

FIG. 1. Acid-urea-polyacrylamide slab gel

electro-phoresis analysis of cellular histones (A, scan of stained gel)andof

[3H]lysine-labeled

histones

pres-entinSV40chromatin(B)andCsClgradient-purified SV40 virions (C). For labeled samples, gels wre

slicedinto 1-mm slices and treated with 0.8 ml of Protosolasdescribed in the text before counting in scintillationfluid.

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355

theHisubfractionand contains highly modified

histones. The most prominent differences

be-tweenthe histones in mature virus and those in viral chromatin are the presence of highly

ace-tylatedforms of histone H4 subfractions and the

relative reduction of the H2a-H2b peak in

ma-ture virus (Fig. 10).The reduction of the H2a-H2b peak in mature virus is not due to the

deficiency of these histones in mature virus,

sincesodiumdodecyl sulfate-polyacrylamide gel

analysis failedto reveal anyquantitative

differ-ences of these histones between mature virus

and viral chromatin (unpublished data). Thus,

theseresults indicatethathistones H2a or H2b

inSV40virion, or both, are modified to a higher extentthanthose in viral orcellularchromatins.

Virion assemblyintermediatesisolated from

su-crosegradientscontain thesame histonepattern

asthat inmature virus except that histone Hi

is present in these complexes. The extensive

modification pattern of virion assembly

inter-mediates as compared with that of viral

chro-matincanbeseenin Fig.2.

Thehigh level of histone acetylation in SV40

virion and virion assembly intermediates

rela-tive tothe viral chromatin wasalso confirmed

by the analysis of thecontent ofE-acetyllysine

inthesecomplexes. Thecontentofe-acetyllysine

inSV40 virions and virion assembly

intermedi-atesis aboutfive times higher than that in SV40

chromatin (Table 1).

Thus, theseresults demonstrate that thereare

twohistone modificationpatterns in

intracellu-larSV40 NPC: (i) aless modifiedpattern

asso-ciated with viral chromatin which is involvedin

thebiosynthesisof viral RNA and DNA(7),and (ii)anextensivelymodifiedpatterninthe virions

and virionassemblyintermediates.

Electrophoretic pattern ofnewly incor-porated histones in SV40NPC. The results

described above show that the histones inSV40

chromatin and virion

assembly

intermediates

aremodifiedtodifferentextents.

Previously

we

have shown that virionassemblyintermediates

arederived from SV40 chromatin

(4,

7). Thus,

the conversion ofSV40 chromatin into virion

assembly intermediates is associated with the

modificationof histones.

However,

it isnotclear

whetherthechangein histonemodification

pat-tem occursearly duringtheassemblyorduring

thematurationofvirions. Toanalyzethis

prob-lem we pulse-labeled SV40-infected CV-1 cells at 48hpostinfectionwith[3H]lysinefor 90 min.

During thisrelativelyshortpulse

period,

labeled

histones wereobserved onlyin

early

virion

as-semblyintermediates(4),asmostof thelabeled

protein couldbedisassociated fromSV40DNA

in theCsClgradient.The histonespresentinthe

.'

-VP3-}_HI

s*# ..4 s.

I f P- .

... ,i4-. .

>-

H3

}

H20

H2b

L

H4-.t~~~~~~~~~~~~~~~~

S_

-I

_0

*ta

1'

Ie

FIG. 2. Acid-urea-polyacrylamide gel

electropho-resis of histonemodificationpatternsin SV40 chro-matin (left column) and SV40 virions and virion precursors (right column) isolatedfrom a sucrose

gradientasdescribedpreviously (6).

pulse-labeled SV40chromatin and virion

assem-blyintermediateswere

analyzed

inacid-urea

gel.

The results are shown in Fig. 3. The labeled

histones which just become detectable in the

newlyformed virion

assembly

intermediate

(150

to 180S in the sucrose gradient) are already

extensively modifiedas inthe maturevirus. In

a separate

experimental

protocol,

the histones

inSV40chromatinwerebriefly pulsedandthen

chased into virionassemblyintermediates. Dur-ing a short pulse (2 to 5 min) labeled histones

were observed in the positions of SV40

chro-matin andreplicationinternediates(5),butno

radioactivityin the positionofvirionassembly

internediates couldbedetected(Fig.4AandB).

During a brief pulse for 2 miin followed

by

a

chase witha 100-foldexcess ofunlabeled

lysine

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TABLE 1. e-Acetyllysinecontentin SV40virion,

virionassemblyintermediates,and viralchromatin'

H3, H2., H2b, e-Acetyl- Lysineas Complex H4(input lysine e-acetyl-cpm) (cpm) lysine

708 chromatin 1.70x 105 2,310 1.36 Assembly interme- 3.84x104 2,737 7.12

diates

Virion 1.98 x104 1,203 6.08

aSV40nucleoproteincomplexeswerelabeled for24

h with[3H]lysineandfractionated in thesucrose gra-dientasdescribedpreviously (7). Analysisof

e-acetyl-lysinewasperformed asdescribedby La Bella et al. (9).

for1h,labeledhistonesbegantobe observed in

the virion assembly intermediates (complete

conversion ofnewlyformedSV40 chromatin into

virionassembly intermediaterequiresatleast4

h of chase, depending on the period of viral

infection). Again, the newly incorporated

his-tones presentin thevirionassembly

intermedi-ates were highly modified as compared with

SV40chromatin (Fig.

4C,

and C3) which is

sim-ilartothesteady-state pattern shown inFig.1.

The pulse-chase experiment also revealed a

subtle change of histone pattern in the newly

synthesized SV40 chromatin. Histone H4 is

more highly acetylated in the briefly pulsed

chromatin than in the chased steady-state

la-beled chromatin (Fig. 4A1-A3). On the other

hand, histone H3 is less modified in thenewly

synthesizedchromatin than inthe chased

chro-matin. These results are similar to those

ob-served incellular chromatin(8).

Analysisof rate of acetylation of histones

in SV40 NPC. The results described above

showthatnewlyincorporated histones inyoung

virionsarealready highly modified. These data,

however, donottelluswhen themodification of

histones in virion assembly intermediates

oc-curs. Modification could occurin afraction of

SV40 chromatin before the accumulation of

cap-sid proteins onto these modified chromatins.

Alternatively,

modification of histones could

happen after the fornation of virion assembly

intermediates. To distinguish these possibilities

we studied the kinetics of one of the major

histone modifications, acetylation, by

pulse-la-beling with

[3H]acetate

(apulse-labeling study

ofhistone phosphorylation was notcarriedout

becauseofthe longequilibration time of the

3P-labeled pool). A short pulse with

[3H]acetate

enablesone tomeasure the rate of acetylation

without concerning theaccumulation of

acety-latedhistones. If the majority of histone

acety-lationoccurs and accumulates in the virion

as-sembly intermediates, then a short pulse with

cY

x

E

0a

u

[image:4.499.57.251.82.185.2]

10 20 30 40

FIG. 3. Acid-urea-polyacrylamide slab gel

analy-sisofhistonespresent inSV40NPCpulse-labeledin vivo with 0.2mCiof[3HJlysinepermlfor90min. The viral NCP wereextracted,fractionatedina sucrose

gradient, andanalyzedin a15%

acid-urea-polyacryl-amide slabgel.(a)SV40virion precursorsedimenting

between 180 and210S; (b) 70SSV40chromatin.

[3H]acetate should label the complex. On the

other hand, ifacetylation of histone occurs in

the 70Schromatin and thenaccumulates in the

virion assembly intermediates, then only 70S

chromatin will be preferentially labeled during

ashortpulsewith[3H]acetate. When

SV40-in-fected CV-1 cells were pulsed briefly with [3H]acetate for10 min,themajorityof histone

acetylationwasobserved in the70SSV40

chro-matin fraction instead ofinthe virionassembly

intermediates (Fig. 5).Alongerlabelingperiod

results in the accumulation of label in virion

assembly intermediates (unpublished data).

Theseresultssuggestthatthe hyperacetylation

of histones in the SV40 virion probably takes

place in SV40 chromatin before the

accumula-tion of capsid proteins. The distribution of

[3H]acetateinthepulse-labeled histonesinSV40

chromatinwasalso analyzedin

acid-urea-poly-acrylamide gel. The result isshown in Fig. 6. As

expected, histone Hi is not labeled. Since

Hi

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0

'1~~~

t14{r

Iiii B

iIilSi1X

B

10 20 304 0203 0 tO 2 04

6

-i

li 1I ilI

BzB3 o haedfo hafera5-inpus with iiifcodyine (C IC3,adtevrlNCwrII

2-~~~~~~~~~~~10

2

II~~~~~~~~~~~2C

FI.w.Pus-caeanalysis of hitn mdfcainprfls fS40NCiacid-urea-plarlmdgelaecie slabntetx.A ftesmls.Sere4ru

simultaneouslywith the addedcellularhistone markers inparallellanes. Thesecellularhistonestandards are shown on top of eachcolumnfor theidentification of individual histone species. Gels were sliced into 1-mmslices and treated with 0.8 ml of Protosol before counting inscintillation fluid.

hasanacetylgroup atits amino terminiadded

during its synthesis in the cytoplasm (13), this

result indicates that themajority oflabels

ob-served in Fig. 5 and 6 are due to the internal

lysine acetylation. The data inFig.5and6show

that histones H3, H4, and H2a in 70S chromatin

arethemajorhistonespeciesacetylatedduring

ashortpulse.

DISCUSSION

Wehavepresented evidencethatthe histone

modification patterns in SV40 chromatin are

strikingly different from those ofSV40 virion

andvirionassemblyintennediates. Whereasthe

histonespresent in thelatter NCPareunusually

highly modified, the SV40 chromatin contains

essentially unmodifiedhistones. Insomeofthe

a

b

c

H.

-I--hi

ll

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H2a

FIG. 5. Autoradiographofhistones inSV40NPCpulse-labeledat 48hpostinfectionfor10min in vivo with

2.5mCiof[3HJacetateper ml.Pulse-labeledproteinsinSV40 NPCwereanalyzedina12%sodiumdodecyl

sulfate-polyacrylamide gelandfluorographed for1monthbythe methodofBonner andLaskey (1). (a) 70S

SV40chromatin; (b) 100Sreplicative intermediate; (c)180to210S virion and virion precursors. Theprotein ontopofeach lanerepresentsanunknownproteinspeciesthatcomigrateswithviralcapsid proteinVP-I.

modificationpatternsbetween thesecomplexes suggest thatchangesof histonemodification

pat-ternsaccompanythetransitionfromSV40 chro-matintovirionassembly intermediates. Our

re-sults with [3H]lysine pulse and chase suggest

that thechanges in histonepatternsoccurearly

during thegenesis of SV40virion.Pulse-labeling experiments with [3H]acetate indicatethatthere

is no major acetylation activity of histones in

thevirionassembly intermediate. Instead,most

of the acetylation activity wasdetected in the

70S SV40 chromatin fraction. This result

sug-gests that the hyperacetylation of histones in virionassembly intermediatesoccursbefore the

accumulation of capsid proteins. Although we

cannot address the question of when histone

phosphorylation mightoccurbecause of

experi-mental difficulties, ourdataof thesteady-state

32P-labeling patternof histones in thedifferent

fractions ofSV40 NCP do not show a

signifi-cantlyhigher degree ofphosphorylation of

his-tonein virionascomparedwithSV40 chromatin (unpublisheddata). These results andour

pre-vious analysis of the SV40 virion maturation

pathway indicate thattransformation of SV40

chromatin into mature virus involves at least

three stages in the following order: (i)

hyper-modification of histones, (ii) accumulation of

capsid proteins, and (iii) organization of capsid

proteinsinto asalt-resistant shell.

The biological function of the unusually

ex-tensive modification of histones in the SV40

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AND HISTONES

H20 Hi H3 H2b H4

I I 1 a.. .

N

0

x

E

10 20 30 40

FIG. 6. Acid-ureagel electrophoresis of

[HJace-tate-pulse-labeled histones in SV40 NPC isolated fromasucrosegradient. Histonesuerepulsed for 10 min with 2.5 mCiof[3HJacetateperml andanalyzed in an acid-urea gel as described in the text. (a)

Assemblyintermediates; (b) 70S SV40chromatin.

virion isunknown. Thepresenceofveryhighly

modified histonesinSV40 virionand virion

as-sembly intermediates butnotinSV40 chromatin

suggests thatmodification of histones is

neces-saryfor theefficient interactionbetween

nucleo-somes and capsid proteins for packaging SV40

DNA-histonecomplexes. Thismaybe the

mech-anism by which the pool of SV40 chromatin

destined for metabolic processes (synthesis of

RNA and DNA) and the pool of chromatin for

the production ofprogenyviruscan be

distin-guished. Thesetwopoolsofchromatinare

pre-sumably equilibrated by the action of histone

modification anddemodification enzymes.The

hypothesisthathypermodification of histones is

necessary for the efficient packaging of SV40 chromatin issupported by the following

obser-vations: (i)nucleosome invirionassembly

inter-mediates remained associated withcapsid struc-turesafter micrococcal nucleasedigestion,

sug-gesting strong interaction between the highly

modified nucleosome and capsid proteins; (ii)

preliminary results of in vitro assembly using

native 70S SV40 chromatin and the 70S

mini-chromosome derived frommaturevirus showed that the latter ismoreefficient inpartial

reas-semblywithcapsids(Coca-PradosandHsu,

un-published data); (iii) host range mutants ofa

relatedvirus, polyoma,aredeficient in modified

histones (14), and the efficiency of virus produc-tion is also suppressed, in spite of apparently normal synthesis of capsid proteins (16). Further

studies are necessary tocharacterize the role of

histone modifications in the interaction between

theSV40 chromatin and capsid proteins for the

formation of SV40 virion.

ACKNOWLEDGMENTS

We thank M.Wilsonand E. M.Johnson for helpful discus-sions and J. Cozzitorto and H.-Y. Yuforexcellenttechnical

asistance.

This work was supported byPublicHealthServicegrant CA-19073 from the National CancerInstitute to M.-T.H. and

bygrantPCM 7907104 from the National Science Foundation toG.V.M.C-P. is a Fellow of the Spanish NationalCouncilfor Research.

ADDENDUM

After thecompletionof thismanuscriptF. LaBella andC. Vesco(J. Virol. 33:1138-1150, 1980) published similar results.

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