Uptake of minute virus of mice into cultured rodent cells.

(1)

0022-538X/79/08-0537/09$02.00/0

Uptake of Minute Virus of Mice into Cultured Rodent Cells

P. LINSER, HELEN BRUNING, ANDR. W. ARMENTROUT*

Departmentof Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati,Ohio45267

Receivedforpublication28December1978

The uptake of minute virus of mice into cells in tissue culture was examined biochemically andbyelectronmicroscopy. Cell-viruscomplexeswere formedat

40C,

anduptake of virus was

followed

after the

cells

wereshifted to 37°C. The infectious particles appeared to enter cells at 370C by a two-step process. The first and rapid phasewasmeasured by the resistance of cell-bound virustoelution by EDTA. The bulk of the bound virus particles became refractory to elution with EDTAwithin 30 min of incubation at370C. The infectious particles became resistant to EDTA elution at the same rate. The second, slower phase of the uptake process was measured by the resistance of infectious particles to neutral-izationby antiserum. This process was complete within 2 h of incubation at370C. During this 2-h period, labeled viral DNA became progressively associated with thenuclear fraction of disrupted cells. The uptake of infectious virus could occur during the G1 phase of the cell cycle and was not an S phase-specific event. The

uptake process was notthe cause of the S phasedependence of minute virus of

micereplication. Inelectron micrographs, virus absorbed to any area of the cell surfaceappearedtobe taken into the cell by pinocytosis.

In many instances, the primary interaction between virus and cell occurs at a specific cell surface component present in limitedquantities (1). These virus receptors may have alimited distribution among the tissues of a susceptible organism, and, insomecases, the receptors ap-pear at specific times during development (3). The tissue specificityof virus infection by polio-virus and the adenopolio-viruses is controlled to some extentbythe presenceorabsence of these virus receptors (1). The nondefective parvoviruses also exhibita degree of cytotropism (6), anda specific virus receptor appearstobeinvolved in infection by theparvovirusminute virus of mice (MVM) (5). The cell receptors for MVM

satu-rate atabout 5 x 105 virus particlesbound per

cell,andthe presence of these saturablebinding

sites has been correlated withsusceptibilityto infection(5). Examination of the cell-virus com-plexes with an electron microscope suggested that the virusreceptor is randomlylocatedon thecell surface (5).

Inthisstudy we examined the early steps in virus infection beyond the initial binding

reac-tion. Afterbinding[3H]thymidine-labeled virus

tocellsat

40C,

we used biochemicaltechniques to measure the synchronous

internalization

of virus at370C. We also examined the uptake of virusby electron microscopic techniques in an attempt to further characterize the early steps oftheinfection.

The replication of the nondefective

parvovi-ruses, includingMVM, requires host cell func-tions whichoccur during S phase (7). It is not clearexactlywhich step in MVMreplication is

subject tothiscellcycle control. Weexamined

the uptake process in synchronized cells and

showed that it is notrestrictedtoS phase but

mayoccurearlyin

Gi.

MATERIALS AND METHODS

Thecelllines used werethe A-9 mouse line and the RT-7ratbraintumorcellline (8). A-9cellsweregrown

inF-liminimalessentialmedium (Grand Island

Bio-logical Co.) supplementedwith 10% heat-inactivated

fetalcalf serum, 100 U ofpenicillinperml,and 100,g

ofstreptomycin perml in suspensionat densities of

from5x 104to 5x 105 cellsperml.RT-7cellswere

growninmonolayersin the above media.Suspensions of mitotic RT-7 cells were prepared as previously describedbymanually shakingculture flasks

contain-ingmonolayersat nearconfluency (8).Cellsprepared

in thisfashionarevirtuallyall in mitosisatthe time of detachment (8).

Virus. Plaque-purified MVM was originally ob-tained from Peter Tattersall. MVM and MVM labeled inits DNA with[methyl-3H]thymidineweregrownin RT-7 cells andpurified as previously described (5). The concentrations ofparticles in the purified viral

preparationswere calculated fromabsorption at280

nm(10).

Binding assay. Binding ofradiolabeled virus to

cellswasperformedinsuspensionasdescribed

previ-ously (5).Briefly,asmall volumeofviruswasadded

tocellsandincubatedat4°C,andthenthesuspension

was filtered through a membrane filter (Nuclepore 537

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

Corp.). The amount of cell-bound virus was deter-minedby measuringtheradioactivityretainedonthe

washed filters by scintillation counting.

Separationof nuclei andcytoplasm.Isolation of nuclei was performed by a modified version of the

method of Wray (11). Cellssuspendedinmediawere

centrifugedat4°Cand2,000rpmfor 5minand

resus-pendedat106cellsperml in buffercontaining50jtM PIPES[piperazine-N,N'-bis(2-ethanesulfonic acid)],1 mM CaCl2, and0.5Mhexylene glycol (2-methyl-2,4-pentanediol; Eastman Organic Chemical Div.,

East-manKodakCo.)atpH6.5. Thesuspensionwas incu-batedat37°Cfor 5 min and thenpassedtwicethrough

a25-gauge needle to burst the swollen cells. Nuclei werepelletedasdescribedabove,and the supernatant

wascollected.The pelletwasresuspendedin 1mlof

isolation buffer and centrifuged at 1,000 rpm. The supernatantsof this and the previouscentrifugation

were combinedasthecytoplasmicfraction. The

nu-clear pellet of the finalcentrifugationwasresuspended

in 1 ml of isolation buffer. These nucleiare90to95%

free of cytoplasmictags(8).Acid-insoluble radioactiv-ityin each fractionwasmeasuredbyprecipitatingthe material with 10% trichloroacetic acid at4°Cfor 1h, using 50,ul ofa 1:10 dilution of fetal calfserum as

carrier. Precipitates were collected by filtration on

WhatmanGFC filters and solubilized with Soluene-100(Packard).Radioactivitywasmeasuredby

scintil-lation spectrophotometry in toluene-based scintilla-tioncocktail.

Electron microscopy. Cells were embedded for

electron microscopy in LuftEpon as previously de-scribed(5).Ultrathin sectionswerecutperpendicular totheplaneofgrowth,mountedon400-meshcopper

grids,and stained withuranylacetateandlead citrate

asdescribed previously (5). Micrographswere taken

with eithera Zeiss EM-9A electronmicroscope ata

40-kVaccelerationvoltageoraJeolJEM 100 B

elec-tronmicroscopeata60-kV accelerationvoltage.

RESULTS

Elution of virus particles from the cell

surfaceby EDTA. We have previously shown

that MVM bound to A-9 cells at 4°C can be

eluted from the cell surface by washing with

Ca2+-Mg2+-free phosphate-buffered saline

(PBS) containing EDTA (5). The earlystagesof

theuptakeprocesswereexamined byfollowing

the elutionof labeled virus fromthecellsurface

using this wash procedure. To observe the

up-take processin thelargest number ofcells,the

cell-virus complexes were formed at 4°C and

subsequently incubatedat37°C (1). Most of the

MVMboundat4°C isatthe cell surface (5).

As Fig. 1 shows, virtually all of the virus

particles bound toA-9 cells at4°C became

re-sistant toEDTAelutionwithin 30min of

incu-bationat37°C.

Elution ofinfectious virus from the cell

surfaceby EDTA. In these virus preparations,

theratio ofinfectious particlestototal particles

ranged from 1:600to1:3,000.Theinteractions of

the labeled particles with the cell surfaces did

a z 0 x 0 49 4c oo0-

50--2 -I I 2 3

HOURS

FIG. 1. Comparison oftherate atwhich cell-bound virus becomes resistanttoelutionbyEDTA with the rate atwhichthe bound particles become resistantto inactivationbyantiserum.Symbols:0,rate atwhich

labeled virus boundtorandomly growingA-9 cells became resistanttoelution byEDTA. A-9cells(106 cells in1mlof serum-freeF-l1medium)werereacted

with [3H]thymidine-labeled virus(2.4 x 1010

parti-cles) for1 hat4°C. Afterthe incubationat4°C,the cellsuspensionswereincubatedat37°C for the indi-cated times. Suspensions were thenfiltered as de-scribed in thetextand washedfor5minat0°Cwith Ca2+-Mg2+-free PBS containing1 mM EDTA. The

cell-associated radioactivity, after cold incubation andwashingwith normalPBS,wasdefinedas100%

(22,500 cpm) for comparison with EDTA-PBS-washed samples. The input quantity ofvirus

con-tainedapproximately23,000cpmofacid-precipitable radioactivity.x,Rateatwhich infectiousvirus bound

tosynchronizedRT- 7 cells became resistanttoelution byEDTA. RT- 7 cellsweresynchronizedand allowed

toattachtoTflasks for2 hat37°C.Themonolayers

wereplacedat4°Candexposedto virusfor2h at

4°C. The cell-viruscomplexeswereshifted to37°C, andatintervals cellsampleswerereleasedfromthe

surface oftheflask with PBS-EDTA solution. The cellswererinsed twice in PBS-EDTAby centrifuga-tion andwerereplated at37C in medium.After2 daysthe cellsweredisrupted,and theamountofviral protein produced was determined by HA titration. The dataarepresentedaspercentagesofthe maxi-mum HA titer obtained (4,096). 0, Rate at which

infectious virus bound to synchronized RT-7 cells became resistanttoneutralizationbyantiserum. Syn-chronized RT-7 cells were allowed to attach to T

flasks and exposed to MVM at 4°C as described above.At intervalsduringthe4°Cabsorption period andduringthesubsequent 37°Cincubation,all

sam-pleswereexposedto19hemagglutination inhibition units ofrabbit anti-MVM antiserum. After 4 h of

exposure to antiserum, themonolayers wererinsed withfreshmedium without antiserumand incubated inantiserum-freemediumfor2days.Thecellcultures

werethenharvested, and theextentof infectionwas

determinedbyHA titration. The dataarepresented

aspercentages ofthe maximum HA titer obtained

(8,192). EDTA I ELUTIONI X ANTI-SERUM I NEUTRALIZATION 0 0 I I

I

I, I 4°1_ 37°1 1 1

J. VIROL.

http://jvi.asm.org/

[image:2.504.266.460.68.248.2]

(3)

UPTAKE OF MVM INTO RODENT CELLS 539

not necessarily reflect the interactions of the

infectious particles. To study the fate of the infectious particles after the cell-virus complexes

were shifted to370C, we examined therate at

which the infectionprocessbecame resistantto

EDTA elution. Inthese experiments,

synchro-nizedRT-7cells wereusedtodetermine whether

uptake of infectious virus couldoccurduring the

G1 phase. RT-7 cells in mitosis had

approxi-mately thesamenumber of viral receptors per

cell(5x105)asrandomly growing A-9cells(Fig.

2).

Synchronized RT-7 cellswereexposedtovirus

at40Candshiftedto370C,andatintervalscell

samples were washed with 1 mM EDTA. This

procedure removed loosely bound virus aswell

as detaching the cells from the surface of the

flask.The cellswerewashed and replated into T

flasksat370C.After 48h, the cell sampleswere

harvested, and the accumulated viral protein

wastitrated by hemagglutination (HA). As Fig.

1shows, therate atwhich thetotal viral particle

population (radioactivity) became resistant to

EDTAafter the shiftto370Cwasapproximately

the same as the rate at which the infectious particles (HA production) became elution resist-ant.

Movement of infectious virus into the

cell. Resistance of bound virus to elutionfrom

the cell surface by EDTA does notnecessarily

result from internalization of the virusparticles.

To measure internalization, we examined the rate atwhich bound virus became resistantto

inactivationby antiserum after incubation of the

cell-viruscomplexesat370C.

When the cell-virus complexeswere exposed

toantiserumbefore incubationat370C,the

cul-tures were infected, but only at a low level, indicating that the bulk of the infectious

parti-cles remainedvulnerabletoneutralizationatthe

cell surface at 40C (Fig. 1). However, after

in-cubationat370C, theinfection processbecame

progressively more resistant to the antiserum

until, after 2 h, the antiserumnolongerinhibited

infection.

The bulk of the virus particles, as well asthe

minor fraction of infectious particles, became

resistanttoEDTAelution fromthe cell surface

atthe same relatively rapidrate. However,

re-sistance ofinfectivitytoantiserum proceededat

aslowerrateandwasmaximal only aftera2-h

incubationofthe cell-viruscomplexesat370C.

The formation ofavirus-receptor complex stable

toEDTAelutionappearedto representarapid

initialstepwhichoccurredat370C before

inter-nalization of theinfectious virus.

Association of viral DNA with the

nu-clear fractionofdisruptedcells.Do the bulk

ofthe virusparticlesenterthe cellatthe same

8-2

2 3 4 5

I.M. x 10-6

FIG. 2. Binding of MVM to mitotic RT-7 cells. A totalof 2 x105RT-7 cellsharvested by mechanical detachment of mitotic cells were reacted with the indicated multiplicities of [3H]thymidine-labeled MVMparticles per cell (input multiplicity[I.M.1)for 2h at4°C in 1 ml of PBS. The bound multiplicity (B.M.) wasdeterminedafter filtration, asdescribed inthe text.

ratethat the infectiousparticles are taken up?

Toanswerthisquestion,wedetermined therate

and extent to which input viral label became

associated with the nuclear fraction of infected

cellsafter in vitrodisruption. Association of viral

label with the nuclear fraction is taken simply

as an indication that the viral particles have

entered the cell. Nuclear association does not indicate whether viral particles have actually penetratedtothe nucleus of the intactcellsnor does it give any information on the physical

state or infectivity of the virus particle which

containsthelabel.Intheseexperiments,the cell-viruscomplexeswereagainformed at4°C; the cellswerethen incubatedat370Ctoallow viral

uptake,andatintervals thecellsweredisrupted

andseparatedinto nuclear and

cytoplasmic

frac-tions. Theproportions of the bound viral label in the nuclear and cytoplasmic fractions were determined from the acid-precipitable radioac-tivity. In randomly growing A-9 cells, roughly 27% of thecell-bound viral label became associ-ated withnuclei after2 hat370C, andfurther increasesoccurredatarelativelyslowrate(Fig. 3).

Wetake the rateofassociation ofvirallabel with the nuclear fraction as

representing

the

rateofentry of theviralparticlesinto the cell.

Theinitial rate of entry of the labeledparticles

was not markedly different from the rate of

internalizationof the infectiousparticles(Fig. 1

and3).

Nucleus-associated viral label isin viral

DNA. In the experiments describedabove,the

bulk of the

[3H]thymidine-labeled

viralmaterial

which wasfoundassociatedwiththecellnuclear

fraction resided in intact single-stranded viral

VOL. 31,1979

http://jvi.asm.org/

[image:3.504.274.434.61.208.2]

(4)

o 60_

0

* 40

2 4 6 8

HOURS AT 370C

FIG. 3. MVM uptake and compartmentqlization in A-9cells. A total of1.4 x 106 A-9 cells in1 mlof serum-free F-li mediumwerereactedwith 4.8x1010

[3HJthymidine-lobeledvirus particles for1h at 4°C.

After the cold incubation, cell suspensions were in-cubated at 37°C with agitation for the indicated times. After thewarm incubation, suspensions were transferred to new siliconized glass tubes at 0°C, and thecells werepelleted. The nuclear and cytoplasmic fractions were isolated as described in the text. Tri-chloroacetic acid-precipitable radioactivity was measuredin the supernatant (unbound) fraction as well as the nuclear and cytoplasmic fractions (see text). Trichloroacetic acid precipitation of the input quantity ofvirus yielded a total of 41,000 to 46,000 cpm. The total radioactivity present in samples for eachtime point(100%) was between38,000and47,000

cpm.Symbols:0, nucleus; 0, cytoplasm.

DNA.A-9cellswereinfected with labeledvirus at40C.Atintervals after incubationat

370C,

the total DNA was extracted from the cells. Over 95%of thecell-bound labelwasrecoveredas16S DNAaftersucrosegradient

centrifugation

(Fig.

4). DNA from each sample was

digested

with

single-strand-specific Si nuclease under

condi-tions which didnotdigestdouble-strandedDNA (9). None of the DNAwasresistantto

SI

diges-tion; the DNAfragments

resulting

from

Si

diges-tionwere5Sorsmallerin size

(Fig.

5).

Thus, at times during the infection when25

to35% of the virallabelwasassociated withthe

nuclearfraction, virtuallyall of the

cell-associ-atedviral label wasin single-stranded DNA of

MVMgenomelength.

Association of viral DNA with the nu-clear fraction of

synchronized

cells. The

associationof viral labelwith thenuclear

frac-tion ofthecellswasusedtoexamine the

move-mentof the bulk of the bound virus into

syn-chronizedcells.AswiththeA-9cells,therateof

associationofvirallabel with the nuclear

frac-tionwasmaximalduringthefirst2hof

incuba-tionat37°C (Fig. 6).

0O HOURS A 3 HOURS B 30

-20

-N 1

6HOURS

0 10 - ~~~~~16S

(~~) tO ~~23S

20 5 S

I10

-10 20 10 20

[image:4.504.77.235.58.225.2]

FRACTION NUMBER

FIG. 4. Sizeof labeled viral DNA recovered from

infectedcells. A-9 cells were exposed to

[3H]thymi-dine-labeled MVM at 4°C for 2 h. The cell-virus complexeswereshiftedto37°C, and at zero time and 3and6htotal cellular nucleic acid was purified by proteaseK digestion and phenol-chloroform extrac-tion. Aportion of each sample was then centrifuged on aneutral15to30%sucrosegradient (SW40, 16 h at25,000rpm). The labeled material was located by scintillation counting a samplefrom each fraction. The sizeof the labeled viral material was determined relative to 3H-labeled Escherichia coli ribosomal RNAruninaparallel gradient (D).

A B

0 HOURS 3 HOURS

44

-cm 2

-0

a 6HOURS

04- ~~~23SA

4

~~~~~55

2

l0

20 10 20

FRACTION NUMBER

FIG. 5. SI nuclease sensitivity of labeled viral DNA recoveredfrom infected cells. A portion of the samples from the experiment described in the legend toFig. 4wasdigested withS nuclease (1 h,

37QC)

and then centrifuged on neutral sucrosegradients (see legendtoFig. 4). Samples of each gradient frac-tion weredirectly assayed for radioactivity by scin-tillation counting. LabeledE. coli ribosomal RNA was runinaparallel gradient (D).

http://jvi.asm.org/

[image:4.504.285.432.64.270.2] [image:4.504.286.435.408.579.2]

(5)

UPTAKE OF MVM INTO RODENT CELLS 541

20

-J

I.-0

U. 0 lo0

2 4 6 8

HOURS AT 37°C

FIG. 6. Accumulation of virallabel in the nucleus of RT-7 cells. A total of 4.4 x 105 mechanically detachedmitotic RT-7cells in 1mlof serum-free

F-11 medium werereactedwith 6.2x 1010 [3Hlthymi-dine-labeled virus particles for1hat4°C.Afterthe

cold incubation, the cellsuspensionsweretransferred

to 25-cm2 Falcon T flasks and incubated for the

indicated times at 37°C. The nuclei were isolated

fromthese culturesasdescribedin thetext,and the

trichloroacetic acid-precipitable nucleus-associated

radioactivitvwasmeasured.

Changes in infectivity of viral particles

duringtheuptakeprocess.Weobserved that

apercentage of the virus boundat40C

sponta-neously eluted from thecellsurfaceat370C.For

example, in theexperiment shown in Fig. 3, 9%

of the input label didnotbindtocellsandwas

found freeinthe supernatant afterabsorptionat

40C. When the cells were incubated at 370C,

17% of the input label was recovered in the

supernatantafter 30min.Between8 and 10% of

thebound virus eluted from the cells when the

temperaturewasraised. The eluted virus

appar-ently remained free from the cell during the

subsequentincubation at370C, asthe

percent-age of unboundvirus remained relatively

con-stant once the temperature was raised. Virus

which elutedat370Cretained itsabilitytobind

tocellsandwas asinfectiousastheoriginalvirus

(data not shown). These results may indicate

that the binding of virus to the cell surface at

40Cdoesnotinactivatetheparticles.

It has been shown that the interaction of

poliovirus andadenovirus withthe cellsurface

at 370C results in loss of infectivity, and this

maybe theinitial stepin theuncoatingprocess

forthese viruses (1, 2, 4). In thecase ofMVM,

we showed that the particles absorbed to the

cellat40Cbecametightly boundwithin 30min

after the cells were shifted to 370C. Does the

formation of this stable cell-virus complex

dis-ruptthe bound viralparticles? As Table1shows,

incubationofthecomplexat370C hadverylittle

effect onthe infectivity of the boundvirus. In

theseexperiments, viruswasrecovered from the

infected cells at intervals up to 4 h after the

complexes were shifted to 370C,when the

up-takeprocess wasessentially complete. The

spe-cific infectivity of therecovered virus decreased

byonlyabout50%, indicating that the formation

ofatight complex with the cells at 370Cdoes not resultin a loss of infectivityfor the bound

particles. Although those experiments did not

necessarily examine the infectious process, the kinetics of inactivation of the bound particles roughly follow the uptake of labeled particles. It

appears that the bound viruses lose infectivity

during or after their internalization. Whether thisloss of infectivity is relatedtoviral uncoating requires further investigation.

Electronmicroscopyof virusuptake.

Be-cause of the low ratio of infectivityto particles

inourMVMpreparations, itwasnotpossibleto distinguish the infectious interactions from the fate of the bulk of the virusparticles by electron microscopy. However, the experiments de-scribed above indicate that there are no gross differences in therate atwhich themassof the virusparticlesenterthecell and therateofentry

TABLE 1. Infectivityof cell-bound virus during the uptakeprocessa

Radioac-

Spe-Infectivity t... cific in- % Spe-Timne_Tune

Ii(ectCIDty

_(TCID50)

toVlty

fectiv- ificin (total ity fectivity cpm)

(Xl104)

5min 6.5x107 5,400 1.2 100 30min 5.6x107 5,900 0.9 75

1h 3.8 x107 5,100 0.8 67

2h 2.0 x 107 3,200 0.6 50

3h 2.8x107 4,000 0.7 58

4h 4.4 x107 8,800 0.5 42

aRT-7

cells

weresynchronizedbymitotic detach-mentandallowed to attachtothesurface of T flasks for2hat37°C.Labeled viruswasabsorbedtocells for 2hat4°C,theinoculumwasremoved,and the mono-layers were rinsedtwice in 4°C serum-free medium

andplacedat370C.Atintervals after the shiftto37°C,

the cells were removed from the surfaces of the T flasks in PBS-EDTA solution, which also removed loosely bound virus. Thecellswerewashed twice in serum-freemedium,resuspended in0.01 MTris,pH 9.0,and frozen. The viruswasextracted from the cells byDouncehomogenization, DNasedigestion (1hat

370C,50,gof DNase perml,1mMMgCl2),and Freon

extraction. Samples of the extracted virus were

as-sayedforradioactivityandinfectivity.The 50% tissue culture infective dose(TCID5o)isthereciprocalofthe dilution of the extracted virus whichgivesapositive HA titer in 50%oftriplicateinfected cultures(5).The

specificinfectivity is theTCID5odividedbythe

acid-precipitablecountsper minutepresentin thesample

ofviral extract used for the TCIDso assays. At the time ofextraction from the infected cells,the input viruspreparation hadaspecificinfectivityof1.1 x104

(108 TCID50per9,100cpm).

0

O

31,

http://jvi.asm.org/

[image:5.504.71.225.60.196.2]

(6)

ofthe infectious particles. On this basis, it re-mainspossible that thecellhandles the uptake ofboth kinds ofparticles in the same fashion, andelectron micrographs of the uptake of the bulk of the particles might also represent the uptakeprocessfor the infectiousparticles.

To visualize the processof virusuptake, A-9

cell monolayers were incubated with

approxi-mately 106 particlesper cell at4°C.The cultures were then incubated at 37°C for 10 min and processed for electronmicroscopy.InFig.7 and

8theuptake of MVM into A-9cells appearsto

occurin severalways. Single

virions,

aswellas

groups of virus, are taken into coated vesicles

viaendocytoticclefts. We have shownpreviously

that MVM binds to these clefts at 40C in the absenceofuptake (5).The clefts bubble into the cell andpinchofftoformfree

cytoplasmic

vesi-cles(Fig. 7).The submembranous

thickening

of

theoriginalcleftnowforms the

rough

coating

of

thevesicle. Thesecoatingvesiclesaregenerally seenclosetothecell surface.

Virus particles are also taken up in smooth vesicles. In Fig. 7a and d and 8a, the virus particles appear to act as multivalent ligands, binding adjacent regions of the

plasma

mem-brane together. Filipodia often become ligated to the cell surface in such a manner. Often a

filipodiumcompletelycoated withvirusappears

tobe internalized bythe cell (Fig. 7d and 8b).

Virus-filled channels running into the

cyto-plasmarefrequently seen (Fig. 8a) and may be the product ofvirus-mediated interconnection ofmembranes. Such channelsmaypinchoff and fuse with other vesicles to form large vacuoles linedon their innersurfaces with viruses. Such large virus-lined vacuoles are frequently seen (Fig. 7 and 8), usually located deeper in the cytoplasm than the coated vesicles. Smaller smooth vesicles containing virusesarealso fre-quently seenatvarying depthsin thecytoplasm (Fig.7and 8).

In Fig. 7b, small vesicles containing single virus particlesare seen.However,intermediate

stages of

single

particle uptake

(monocytosis)

werenotobserved,andthese vesicles with

single

virusparticlescould representcrosssectionsof thechannelsofvirusdescribed above. The fact

that activemonocytosiswasnotobserved may

be duetothe

difficulty

ofaccurately

identifying

single

particlesofthis sizeatthecell surface.

Double-walled,

smoothvesicleswereoften

ob-served. These vesicles appearedtobe channels

ofviruswhich had circularized.The central

re-gion

of suchvesicles couldeitherbeempty,asa

vacuole, orenclose amatrixverysimilartothe

surrounding cytoplasm (Fig.8b and

e).

When virusbindinganduptakewereallowed

toproceed simultaneously at 370C, the uptake

process was notnoticeably different from that observedwithpriorviralabsorptionat40C

(data

not

shown).

UninfectedA-9 cells wereembeddedin

mono-layersandsectionedperpendiculartotheplane

ofgrowth(datanotshown).Thecellsurfacewas

clear ofparticulates. Theplasmamembraneof

these cells frequently exhibited thickened

re-gions which appeared to give rise to coated

vesicles. In the absence ofvirus these regions andvesicleswerefreeofparticles.

DISCUSSION

In this study, we examined some aspects of

the uptake of MVM by cultured rodent cells.

Theuptakeoftheinfectious particlesappearsto occurbyatwo-step process. The majorityof the

particles,aswellastheinfectious particles,first

form an EDTA-resistant complex at the cell

surface at 370C. Formationofthis initial

com-plex is relatively rapid and is complete by 30 min.Theinfectious particles then leave thecell

surface and become resistant to antiserum

in-activationat a slower rate. Infectiousparticles leavethe cell surfaceatapproximatelythesame ratethat viral labelbecomesassociated withthe nuclearfractionofdisrupted infected cells. From

thesedata,theinfectious particlesappear to be

FIG. 7. MVM uptakebyA-9cells. ViruswasappliedtoA-9cells(106particlesper cell) in amonolayerat

4°Cfor1h.Approximately2 x 105particleswereboundpercell, asmeasuredby aparallel binding assay.

Thecultureswereincubatedat37°Cfor 10mintoallow viral uptakeandwere thenprocessedforelectron

microscopy. (a)Arrowsindicatestages of virus uptake into coatedvesiclesfrominvagination(right and left)

toautonomous vesicle (center).Arrowheads show patchesofviruscontinuouswithfilipodialandflat surface plasma membrane. x65,000. (b) Aportionof acoated vesiclecontaining virus (arrow)isseenadjacentto an apparentmonoendocytotic vesicle (arrowhead). Crossed arrows indicatealarge, smooth vesicle coatedon its inner surface with virusand asmaller, smoothvesicle onlypartially coatedon itsinner surface with virus.

x54,000. (c) Small arrow indicates a coated vesicle containing virus fairly deep in the cytoplasm. This

representsasdeepapenetrationof thecytoplasmaswaseverobservedforcoatedvesicles. Crossed arrows indicatealarge vesicle andasmaller, smoothvesiclethat mayactuallybecontinuous with each other.The largearrow atthebottom (alsoseeinset)indicatesasmooth vesicle stillin theprocessofinvagination. Virus completely fills the resultant vesicle. Virus in a patch is also evident at the cellsurface(arrowhead).x59,000.

(d) Afilipodium, apparently coated with virus. Thefilipodiumseems to be bound to the adjacent surface

membrane via itscoating ofvirus. x68,000.

J. VIROL.

http://jvi.asm.org/

(7)

INTO RODENT CELLS 543

2a.

1..

zol*-

A-W`-s* - 4,

X.v;St Aas ..-dta ~

.d At

4 I

.-W

k

.404I;**.. I

w

*-;4,.

6A

http://jvi.asm.org/

(8)

Atrt 7 '~aor~ V

O.

-.7

"

'4

FIG. 8. MVMuptake intoA-9icells. The experimental conditions are the same as those describedin the legend to Fig. 7.(a)Arrows indicate severalplaces where channels of virus have been formed by the apposition of membrane surfaces. Filipodia are involved in some of these. x30,000. (b) Arrows indicate several large vesicles with smooth surfacescontainingnumerous virus particles. One suchvesicle(center)appearsto consist of two membrane surfacesinterconnectedwithvirus,forming acircularchannel. The crossed arrowindicates a smallpatch of virus that has not been internalized. The arrowhead indicates a coated vesicle possibly containinga singlevirusparticle. x42,XX0.(c)Arrow indicates a circularchannelof membrane-bound virus. This may be a cross sectionthroughthe base of a filipodium that is sunken into thecytoplasm.Arrowheads indicateviruses that have not yet beeninternalized. x80,OOO.

544

http://jvi.asm.org/

[image:8.504.61.456.40.591.2]

(9)

taken into the cells asrapidly as the bulk of the

viral particles whichdo not initiate infection.

The uptake of infectious virus occurs in the earlyG1 phase of the cell cycle and is not limited to the S phase. Therefore, neither the binding nor the uptake of virus is the cause of the S phase restriction of MVM replication.

Spontaneous elution of virus from the cell surface has been observed with adenoviruses (1) and picornaviruses (2, 4). In these cases, virus bound to the cells at40Candeluted at370C is no longer capable of binding again to the cell due to somephysical change in the viral particles (1, 2). Approximately 8 to 10% of the MVM

particles boundtoA-9cellsat4°C eluteat37°C

and remain unbound even after an additional 8 h of incubation at 370C. However, binding of parvovirus to cells at 40C does not appear to alter theparticles in any significant way, as the eluted virus remains fully infectious. In addition, the formation of the initial stablecell-virus com-plexes at370Cdoes notdisrupt the bound virus. Virtually all of the bound virus has formed an EDTA-resistant complex within 30 min at370C, and theviruses recovered from thecellsatthis time have lost little of their infectivity. On the other hand, there is a progressive decline in the infectivity of virus recovered from cells during

theinternalizationprocess. It remains to be seen

whether this loss ofinfectivity in the particles taken into the cells canbe relatedto an infec-tiousuncoatingprocess.

ACKNOWLEDGMENTS

The assistanceofRandyRichards intheanalysisofviral

DNA isgratefully acknowledged.

Thisinvestigation was supported by Public Health Service grants 1K04 CA-00134 and5RO1 CA-16517from the Na-tionalCancer Institute and by grant 1-396 from the National Foundation March ofDimes.

lTERATURE CITED

1.Dales, S. 1973. Early events in cell-animal virus interac-tions.Bacteriol. Rev. 37:103-135.

2. Fenwick,M. L,and P. D.Cooper.1962.Early interac-tions betweenpolio virus and ERKcells: some obser-vations on the nature and significance ofthe rejected particles. Virology 18:212-223.

3. Holiand,J. J. 1961.Receptor affinities as major deter-minants ofenterovirus tissue tropism in the human. Virology15:312-326.

4. Joklik, W. K., and J.E.Darnell,Jr.1961.The absorp-tion and early fateofpurifiedpoliovirusinHeLacells. Virology 13:439-447.

5. Linser, P. L.,H. Bruning, and R. W.Armentrout. 1977. Specific binding sites for aparvovirus, minute virus of mice, on cultured mousecells. J. Virol. 24:211-221.

6. Lipton, H.L.,and R. T. Johnson.1972.The pathogen-esisof rat virus infections in the newborn hamster. Lab. Invest. 27:508-516.

7.Rhode,S. L., m. 1973.Replicationprocess ofthe parvo-virus H-1. I. Kinetics in aparasynchronous cell system. J.Virol. 11:856-861.

8. Richards, R.,P.Linser,and R. W. Armentrout.1977. Thekinetics of assembly of a parvovirus, minute virus ofmice, insynchronized rat brain cells. J. Virol. 22: 778-793.

9. Sutton,W. D. 1971. A crude nucleasepreparationsuitable for use inDNA reassociation experiments. Biochim. Biophys. Acta 240:522-531.

10. Tattersall, P.,P.J.Cawte,A. J.Shatkin,and D.C. Ward. 1976.Three structuralpolypeptidescoded for by minute virus ofmice,aparvovirus.J.Virol. 20:273-289.

11. Wray,W. 1975. Parallelisolationproceduresfor meta-phase chromosomes, mitotic apparatus, and nuclei. MethodsEnzymol.40:75-89.

31,

http://jvi.asm.org/