Copyrighti 1974 AmericanSociety forMicrobiology Printed inU.S.A.
Involvement
of
the Host Cell Nuclear
Envelope
Membranes
in
the
Replication
of
Japanese Encephalitis
Virus
E. ZEBOVITZ, J. K. L. LEONG, ANDS. C. DOUGHTY
Naval Medical Research Institute, Bethesda, Maryland 20014 Received forpublication 1March1974
The distribution of viral ribonucleic acid (RNA) on various cell membrane fractions derived from a porcine kidney cell line infected with Japanese encephalitis viruswasinvestigated. At 40 h postinfection, after virus growth had reached itspeak, three viral RNAs, 45S, 27S, and20S, wereassociated withthe cytoplasmic membranes and intact nuclei. TheamountofeachRNAassociated with the nucleus was two- to fivefold greater than that present with the cytoplasmic membranes. Treatment of washed infected nuclei with 1.0%Triton X-100, which removed theouternuclear envelope membrane, also removed the viralRNA. When the nucleuswasfractionated into nuclearenvelope membranes andalarge particle fraction which sedimented at600 x g, nearly all of the viral
RNA remained associated with the envelope membranes. Thenuclear envelope membranes contained higher viral RNA polymerase activity than the cytoplas-mic membranesderived from thesamecells.These datasuggestthatmajor sites for Japanese encephalitis virus RNA synthesis may be localized on orin very close association with the nuclear envelope membranes.
Currentconcepts ofviralreplicationvisualize
the cytoplasmic membranes of the host cell as
the site of synthesis and assembly of various
viralcomponents (1, 2, 4-6,8,9, 17).Inthecase
of the small ribonucleic acid RNA viruses, it
appears that the cell nucleus plays little or no
direct role in viral replication. However, for a
wide variety of viral agents such as tobacco
mosaic virus (11, 19), encephalomyocarditis
virus (3, 7, 16), and influenza virus (13), the
possibility that the nucleus has an important
function in RNA replication has been
sug-gested.
In a previousstudy (24), wedescribedseveral
virus-induced RNAspecies associated with
Jap-anese encephalitis virus (JEV)-infected cyto-plasmic membranes which were not found in uninfected cells. They were the 45S and 27S
single-stranded forms and a 20S
double-stranded RNA. In this paper, we present evi-dence whichsuggeststhata majorproportionof
the viral RNA found in JEV-infected cells is
synthesized either in close association with the
host cell nucleus or on the nuclear envelope membranes.
MATERIALS AND METHODS
Cells. Aporcine kidney stable celllinedesignated
as PS(Y-15) (12), cultivated as described previously
(24), was used. Cell monolayers were prepared in 60-mmtissuecultureplates.
Infection and labeling of viral RNA. A 10%
mousebrain suspension (1.0to3.0 x108 plaque-form-ingunits [PFUVml)oftheNakayama strain of JEV servedasthe virus seed. Therelativelylow virustiters
ofseed stocks didnotpermit the infectionofcells with highmultiplicities. However, in several preliminary experiments where cultures were infected with ap-proximately 10 PFU percell, the growth responsewas
similarto that obtained with multiplicities of1 to5
used routinely in these studies. The virus was ad-sorbedfor 1hat 35C, and the cultureswereoverlayed with 5 mlofHankslactalbumin hydrolysate medium (Grand Island Biological Co.) supplemented with10%
heat-inactivated calf serum. The cultures were incu-bated at 35C in an incubator supplied with 5.0% carbon dioxidein air.
ReplicationofJEVcomparedwith that ofgroupA
arboviruses is slow. Virus titers of JEV grown on
PS(Y-15) cells begin to increase at 10 h and reach maximaltitersrangingbetween 106 to 107PFU/mlat 33 h. In most experiments described here, virus
samples were taken during early (16 h) and late (40 h) growth stages. Inseveral experiments where sam-ples were taken at 33 h, sucrose density gradient sedimentationpatterns ofviral RNAwere similar to those at 40 h. JEV replication is inhibited by ac-tinomycin D (24). The drug, therefore, could not be addedatthetimeofinfection to restrict cellular RNA synthesis. Tolabel viral RNA,1Agofactinomycin D perml wasadded with5 ,uCiof [5-3Hjuridineper ml
atthe appropriate time postinfectionand was
reincu-204
on February 25, 2021 by guest
http://iai.asm.org/
bated at 35 C for the desired interval. Under these ditions, host cell RNAsynthesis wassufficiently de-pressed to detect viral RNAproduction.
Cellswere harvested by scraping into the overlay medium with a rubber policeman and were washed
twice by centrifugation with phosphate-buffered saline, pH7.4(PBS).
Preparation ofcytoplasmic membrane and
nu-clear fractions. Procedures forthelysisofcellsand isolation of cytoplasmic membrane fractions were
described previously (24). The nuclear fraction
re-maining after the removal of the cytoplasmic
mem-braneswas subjectedto aseriesofwashesto remove
cytoplasmic contamination (23). The nuclearfraction
wassuspendedby meansofaDounce homogenizerin
10volumesof2.3Msucrosecontaining 1.5 x 10-3 M CaCl,and10-2Mtris(hydroxymethyl)aminomethane (Tris)buffer(pH 7.5). The nucleiweresedimentedby centrifugation at35,000xgfor 30min.Thepelletwas
washed by resuspension in3 to 4volumesof 0.23 M sucrose containing 3.0 x 10-3 M CaCl, and
cen-trifugedat 600 x g for5 min.Microscope examination
of the nuclear pellet indicated that it consisted primarilyof intactnuclei with alowlevelof particu-late contamination. It wasnot possible to determine whether the contaminatingmaterial was cytoplasmic or due to the lysis of somenuclei.
Nuclear envelope membranes were obtained by disrupting the washed cell nuclei with a Dounce homogenizer after allowing them to swell for 30 minin 10volumes of 2.0 x 10-2 Mphosphate buffer, pH 7.2 (23). The lysed nuclear suspension was centrifuged at
600 x g to remove intact nuclei and large-particle nuclear debris. The supernatantfluid containing the nuclear envelope was centrifugedat47,000 x gfor 20 min.
For the determination of the incorporation of
['H]uridine by cell membranes, cytoplasmic and nuclear envelope membranes from uridine-labeled cellsweresuspendedin 5mlofreticulocyte standard buffer (0.01 MTris-hydrochloride,0.01MKCl, 1.5 x
10 'M MgCl2), and 1-mlsamples were removed and
precipitated with 10% trichloroacetic acid.
Precipi-tates werecollectedonmembrane filters, and radioac-tivitywas measured.
RNA extraction. A cold phenol-sodium dodecyl
sulfate procedure wasusedtoextract RNA from the cytoplasmic and nuclear fractionsasdescribed previ-ously (24).
Sucrose density gradient analysis. RNA samples
inPBS were layered on 15 to 35% sucrose gradients andcentrifugedfor 18hinanSW-25.1 rotor at 23,000 rpm in a model L Spinco centrifuge. Chick cell ribosomal RNA was added to provide 18S and 28S ribosomal RNA as sedimentation markers. Tubes were pierced from the bottom and 10-drop fractions were collected. Acid-insolubleradioactivity was
mea-suredby precipitating the fractions with10%
trichlo-roacetic acid. Precipitates were collected on 3.0-gm
membrane filters which were dried and placedin 10
mlof aliquid scintillator solution (4.0 gofOmnifluor [New England Nuclear] in 1 liter of toluene) and
counted with a Beckman LS-250 liquid scintillation spectrometer.
Forthe determination ofribonuclease resistance of viral RNA, the volumes of sucrose density gradient fractionswereadjusted to 2 ml withPBS and divided
into two 1-ml portions. One set was processed for acid-precipitable RNA without further treatment. Ribonuclease (1Mg) was added to each fractionofthe second set, and the samples were incubated at 37 C
for 30 min, treated with trichloroacetic acid, and processed asdescribed above.
RNA polymerase activity. The procedure de-scribed by Plagemann and Swim (18) was used to assay for RNA polymerase activity associated with cell membrane fractions. The reaction mixture
con-sisted ofTris-hydrochloride buffer (pH 8.2), 50 MM; sodiumphosphoenolpyruvate,4MM; magnesium
ace-tate, 5MM;adenosine, cytidine, and uridine triphos-phates, 0.06MM each; actinomycinD, 10Mg; pyruvate kinase, 10g; and [3H]guanosine triphosphate, 1MCi.
A 0.5-ml amount of cell membrane suspension pre-pared from normal or infected cellswasadded tothe reaction mixture to a total volume of 1.0 ml. The reaction mixture wasincubated for 30 min at 37 C.A
1-ml amountof0.1 M sodium pyrophosphate contain-ing 2 mg ofbovine serum albumin and 2.5 ml of 1M
perchloric acid containing 25 mg ofkieselguhr were added tostopthereaction.The resultant precipitate was washed twiceby centrifugation with 5 ml of 0.5 M perchloric acid and once with 5ml of0.5M trichloro-acetic acid. The pelletwas resuspended in 1.0ml of 0.5Mtrichloroacetic acid and heated in a70-C water bathfor30 min.The precipitate was transferredto 20
mlof adioxane-based scintillation fluid, and radioac-tivity wasmeasured.
Protein content of the cell membranes was deter-minedby the Lowry method (15), withbovine serum albumin fractionVasthe protein standard.
RESULTS
Viral RNA species associated with the
cytoplasmic membranes and nuclear
frac-tion. Figure 1 compares the sucrose density sedimentation patterns of RNA extracted from
cytoplasmicmembranes and washed intact
nu-cleiat 16and 40h. Cellswere incubated inthe presence of[5- 'H]uridineandactinomycin D for 2 h before isolation of the cytoplasmic and nuclear fractions. Viral RNA synthetic activity was detected in JEV-infected cells at 16 h as well as at 40 h postinfection (Fig. 1). The distribution of viral RNA species between the
cytoplasmic membranes and nuclear fraction
appeared to be different at the 16-h stage of
virus replication. The 27S and 20S viral RNA-forms were detected on the cytoplasmic
mem-branes, but a 45S RNA peak wasnot seen. On
the otherhand, 45SRNAwaspresent as asmall but discrete peak associated with the nuclear fraction. At 40 h, both cellular fractions
con-tained 45S, 27S, and 20S viral RNA species.
The nuclear fraction, however, was found to
contain two- to fivefold more viral RNA than
on February 25, 2021 by guest
http://iai.asm.org/
ZEBOVITZ, LEONG, AND DOUGHTY that associated with the cytoplasmic
mem-branes. The 45S and 20S species were consist-ently predominant, but 27S RNA, shown as a shoulder in Fig. 1, was always a minor compo-nent.
The sedimentation pattern of RNA extracted from the nuclear fraction of uninfected cells is
comparedwith that ofinfected cells in Fig. 2. At
16 and 40 h postinfection, a single prominent
RNA with a sedimentation coefficient of
ap-proximately 4S was found in uninfected nuclear fractions, whereas the45S, 27S, and 20SRNA forms were present only in RNA extracts of
16Hr x D z U) z 0 Q, 0 28S 18S 4S 27S __
infected nuclei. In addition, the 20S RNA was resistant toribonuclease and
presumably
repre-sents the viral double-stranded RNAs(replica-tive form and replicative intermediate)(Fig. 2).
These data strongly suggest thatthe 45S, 27S,
and20S RNAsfound in infected cellswerevirus
induced. Similar resultswereobtainedforRNA
associated with infected cytoplasmic
mem-branes (24).
The 4S RNA was found associated with the
host cell nucleus of both normal and
virus-infected cells, but largeramounts were
consist-ently formed in infected nuclei. This RNA
40 Hr
0 10 20 30 40 0 10 20 30 40
FRACTION
FIG. 1. Sucrose density gradient sedimentation profiles of RNA extracted fromcytoplasmicmembranes and intactnuclei derivedfromPS(Y-15)cellsinfectedwithJEVfor16and40h.Cytoplasmicmembranes,0;nuclei,
*. 5r 16Hrs (9 z
2L1
o 0, 28 S 18S14
4S 0 10 20 30 40 0 10 20 30 40 FRACTIONFIG. 2. Sucrose density gradientsedimentationpatterns of RNA extracted from nuclear fractions derived from uninfectedandJEV-infected (PS-Y15) cells. Uninfected, A; infected, 0;infected and treated withI
gg
of ribonuclease for 30 min at 37 C, A.206
on February 25, 2021 by guest
http://iai.asm.org/
species seems to be a host cell product and its relationship, if any, to JEV RNA synthesis is notknown.
Viral RNA synthesis associated with the nuclear envelope. The nuclear envelope is composed of two membrane layers. The outer
membrane is directly connected to the
endo-plasmic reticulum and is usually regarded as a part of it. The inner membrane is a
nuclear-derivedstructure (23).
When isolated nuclei are treated with non-ionic detergents such as 1% Triton X-100, the outer membrane is removed, leaving the nuclei intact (22). By exploiting the property of non-ionic detergents to selectively remove the outer nuclear membrane, we attempted to determine whether viral RNA found on the nucleus was associated with nuclear envelope membranes.
Asuspension of washed nuclei obtained from 40-h infected cells was divided into two equal portions. One served as the untreated control
and was processed for the extraction of RNA.
The second sample of nuclei was washed once with 1.0% Triton X-100 before RNA extraction.
Microscope examination of the
detergent-washed nuclei indicated that they were still
largely intact. TheRNA sedimentation pattern
ofboth samples is shown in Fig. 3. Treatment with 1.0% Triton X-100 removed virtually all of
the viral RNA originally associated with the intact infected nucleus, indicating that the RNA may have been localized on the outer
membranes.
To further establish the location of the viral
RNA associated with the nucleus, washed
in-tact nuclei obtained from 40-h infected cells
were ruptured and the nuclear envelope
mem-branes were isolated. The sedimentation
pat-terns ofRNA extracted from the nuclear
mem-branes and from the heavy particles of the
nucleussedimentedby low-speedcentrifugation (600 x g) are shown inFig. 4. The45S and 20S viral RNA species were associated primarily with the nuclear envelope membranes. The
large-particle nuclearfraction contained no
de-monstrable amounts of45S RNA, very little, if
any, 20SRNA, and almost allthe 4S RNA. The association of viral RNA with the nuclear envelope membranes suggests that the sites of viral RNA synthesis are localized on these membranes. It is conceivable, however, that viral RNA synthesis takes place primarily on
the cytoplasmic membranes, but during the
long periods ofcontact (2 h) with [3H]uridine
used in above-described experiments, viral
RNA products were eventually transferred to
the nuclear envelope membranes. To test this
4S CY N z a z 0 FRACTION
FIG. 3. Effect of Triton X-100 upon RNA associ-ated with intact nuclei derived from JEV-infected cells. Untreatednuclei, 0; Triton X-100-washed
nu-clei,0.
possibility, cells infected for 33 or 40 h were
labeled with [3H ]uridine in the presence of
actinomycin D for 5, 10, or 30 min. Cells were
fractionatedintocytoplasmic and nuclear
enve-lope membranes, and the specific radioactivity
of each fraction was determined. Variation in
the amount of radioactivity incorporated
by
each membrane fraction was encountered
among experiments (Table 1). The nuclear
envelopemembranes, however,incorporated 1.4
to 6.6 times as much as thecytoplasmic
mem-branes derived from the same cells. Reducing
the pulse interval did not changethe
distribu-tion of the labeled uridine in favor of the
cytoplasmic membranes.
Todetermine whether theradioactivity mea-sured in the short pulses
(Table
1) indeedrepresented viral RNA, the following
experi-ment was
performed.
RNAwas extracted fromcytoplasmic membranes and from washed
in-tact nuclei labeled with [3H
Juridine
for 10 min at 33 h postinfection and fractionated on su-crose densitygradients(Fig.
5). The viral RNA 207on February 25, 2021 by guest
http://iai.asm.org/
LEONG,
-I
"4
cytoplasmic membranes. An RNAspecies
withx', a sedimentation coefficient of
33S,
not seen inl0 previousexperiments, wasobservedas a
promi-nentpeak on the nuclear fraction. It
might
be9 925S2155lI Sl analogous to the RNA form describedNAfr dsrbd
by
TLevinN
8
/
and Friedman (14) for groupA
arboviruses._ 8
Corresponding
sucrosedensity gradient
RNAw x, , profiles of uninfected PS(Y-15) cytoplasmic
membranes and nuclei did not have the viral
Z 6 _ RNA
peaks
described above.E x x Viral RNA
polymerase activity
associatedsX 5 with cellmembranes. The distributionofviral
z
o 45S x RNA polymerase on various cell fractions was
84
- 20S examined intheexperiments
described below.Cytoplasmic membranes and washed intact
3 - /x nuclei derived from normal and 40-h infected
lw x-
*
PS(Y-15)
cells weretested forRNApolymerase
2 _-J XJFxx-e %t activity. The resultant RNA products were
extracted with
phenol
andanalyzed
onsucroseI xxx,oxi density gradients (Fig. 6). The major RNA
spe-0 I ,I cies formed
by
the JEV-infectedcytoplasmic
0 10 20 30 40 membrane and nuclear fractions in vitro had a
FRACTION sedimentation coefficient of 20S and was
resist-ant to ribonuclease.
Corresponding
fractionsFIG. 4. Distribution of viral RNA on
the
nuclear from uninfected cells did not synthesize this envelope membranes and large-particle nuclear de- RNA species in vitro. Thus the 20S RNA bris. Nuclear envelope membranes, 0; large-particle appears to be a virus-induced double-stranded nuclearfraction, x.TABLE 1. Incorporation of[5-3H]uridineinto cytoplasmic and nuclearenvelopemembranesof
JEV-infected cells
Spacta Periodof o
pulse No.
ofC
Nuclear Ratio5(m) expts Cytoplasmc envelope
membranes 5 3 1,164 3,793 3.2 160 914 5.7 12,140 38,000 3.2 10 5 1,520 6,331 4.2 1,930 12,850 6.6 29,300 48,000 1.6 3,049 4,248 1.4 14,485c 20,558c 1.4 30 1 4,500 13,550 3.0
aBasedonthreereplicatesamplesandcorrectedby
subtracting the specific activityof uninfected
mem-branes.Expressedascountsper minute permilligram
ofprotein.
'Ratio = (specific activity of nuclear envelope membranes)/(specific activity of cytoplasmic
mem-branes).
eSamplesweretaken at33 hpostinfection.
speciesdescribedearlier(Fig. 1)werepresenton
bothcellfractions, butthe amount of each RNA
associated with the nuclearfraction was three-to fourfold greater than that present on the
30 r 25 I cli N
I0
x w z -z 0 0 20 V 15 33S 20S 45S I0 I 5 o 0 10 20 FRACTION 4S 2jS ass 'O'bo~~~~~~~~~~'/~~~~W
9~~~~~~ 0 30 40FIG. 5. Viral RNAs associated with the cytoplas-mic membranes and nuclearfraction aftera 10-min pulse with [5-H]uridine. Nuclearfraction, 0; cyto-plasmicmembranes, 0. 208 INFECT.IMMUNrrY ALI
on February 25, 2021 by guest
http://iai.asm.org/
Downloaded from
VOL.10,1974 JEVRNASYNTHESIS ON NUCLEARENVELOPE MEMBRANES 250k A 28S 18S 200F L,. D z 1-50 2 _ z 0 Q) I
L
20S -0 10 20 30 40 0 10 20 30 40 FRACTIONFIG. 6. Sucrose density gradients of RNA synthesized by RNA polymerase associated with cytoplasmic
membranes and with washed intact nuclei derived from normal and JEV-infected PS(Y-15) cells. A,
Cytoplasmic membranes; B, nuclear fraction. Infected, 0; uninfected, 0; infectedand treated with 1 migof
ribonucleasefor 30minat37C, A.
RNA. Other viral RNA forms seemed to be formed by viral RNA polymerase in vitro but were not clearly resolved on sucrose gradients (Fig. 6).
Largeamountsof 4S RNAwerealso formedin vitro by nuclei derived from normal and virus-infected cells. These results support ourearlier observation (Fig. 1) that the 4S RNA is proba-bly notvirus induced.
Our studies have suggested that viral RNA synthesis can take place on cytoplasmic and nuclear envelope membranes, but the latter appeared toplay amore significant role. If the major site of viral RNA synthesisoccursatorin close association with the nuclear envelope membranes, it would be expected that these membranes would have a higher level ofRNA polymeraseactivity than the cytoplasmic mem-branes.Thiswastestedinthenextexperiment.
Cytoplasmic and nuclear envelope mem-branes derived from 40-h infected cells were tested for RNA polymerase activity. Since the yield of nuclear envelope membraneswas gener-ally much smaller than thatofthecytoplasmic membranes, the values were corrected on the basis ofprotein content(counts perminuteper
milligram of protein). Tocorrectforthe contri-bution of residual actinomycin D-resistant cel-lularRNApolymerase activity, the correspond-ing activity of uninfected cell membranes was subtracted (Table 2).
The nuclear envelope membranes exhibited 1.7-and4.3-foldgreaterRNApolymerase activ-ity than did cytoplasmic membranes from the
TABLE 2. Distribution of viral RNA polvmerase activitv oncvtoplasmic and nuclear envelope
membranes
Spacta
Expt Cvtoplasmic Nuclear Ratiob
. ~~envelope
membranes membranesevlp
1 3,314 14,358 4.3
2 483 823 1.7
a
Specific
activity
wascorrected forRNA polymer-aseactivity present in corresponding membranes of uninfected cells.Expressed as in Table 1.bExpressed as inTable 1.
same cells. This observation appeared to
indi-catethatthe membranes associated with the in-fected cellnucleushad ahigher specific activity
ofviral RNA polymerase than the cytoplasmic membrane.
DISCUSSION
Evidenceofnuclearinvolvement in JEV RNA
replication wasobtained byTakeda etal. (20),
who demonstrated viral RNA association with
the nucleusby radioautographic techniquesand
by Takehara (21), whoreported that 15 to 30% ofthe total RNA polymerase found in infected
Verocells resided in the nuclear fraction. A special role of the nucleus in the synthesis of viral RNA by group B arboviruses was indicatedbyBrawner et al. (Abstr. Annu. Meet. Amer. Soc. Microbiology, 1973, p. 202). They
found that viral RNA was localized on the 209
on February 25, 2021 by guest
http://iai.asm.org/
ZEBOVITZ, LEONG,
nucleus, and theydetectedindetergent-washed
nuclei a45S viral RNAenriched in
polyadenyl-ate [poly(A)
].
They suggested that a nuclearfunctionmaybe involved inviral RNA
process-ingandinthe additionofpoly(A)to45S RNA.
Our initialexperiment(Fig. 1) indicated that
cytoplasmic membranes and nuclear fractions
wereinvolvedinthesynthesisofviral RNA but
that a major proportion of the virus-induced
RNA was associated with the nuclear fraction.
Subsequent experiments (Tables 1 and2)
sug-gested that this association reflected thesiteof
viral-induced RNAsynthesis. Theratio ofRNA
associated with the nuclear fraction,ornuclear
membrane,
tothatrecoveredinthecytoplasmic
membrane fraction did not materially change
when the pulse of exposure to labeled uridine
was changed from 2 h to 5 or 10 min; in
addition, viral RNA polymerase activity was
higher in the nuclear envelope fraction than in
the cytoplasmic membranes.
The possibility that the radioactivity and
RNA polymerase activity associated with
in-fected cell nuclei and nuclear envelope
mem-braneswasduetocontamination with
cytoplas-micconstituentswasconsidered. The procedure
forthe isolationofcell nucleiprobably doesnot
totally exclude cytoplasmicelements, but
light
and electron microscopic examination of the
nuclear preparations revealed the presence of
limited cytoplasmic material. To account for
the level of RNA synthesis shown in Fig. 1, 5,
and6, it is necessary to assumethatournuclear
preparations were grossly contaminated with
cytoplasmic
constituentsorthat theseconstitu-ents had aremarkably high specific activity.
The nature of the 4S RNA found in large
amounts associated with JEV-infected nuclei
(Fig. 1-6) is not known. Control uninfected
nuclear fractions contained this RNA
species,
but infection substantially increased its level. In apreviousstudy(24), we found that
actinomy-cin D inhibited ribosomal RNA synthesis of
uninfected PS(Y-15) cells but increased the 4S
RNAcontent. Actinomycin D is known to cause a conversion of nuclear RNA to acid-soluble
products (10). One possible explanation of the
large amount of 4S RNA is that it represents
cellular and, perhaps, degraded viral RNA
re-sultingfromthe action ofactinomycin D.
The results shown in Fig. 3 and 4 provide indirect evidence that the outer layer of the nuclear envelope may be the site of virus-induced RNA synthesis. The nuclear envelope
membrane contained nearly all the recovered
radioactivity associated with the viral RNA
species, whereas the large nuclear fractions
containedalmostexclusively 4SRNA; however,
when the outernuclear envelope was removed
with detergent, the viral RNA was also
re-moved.
The relationship between RNAsynthesis
as-sociated with the cytoplasmic membrane to
that foundon the nucleus is not known. Early
(16h)inthevirusinfection, we detected the45S
viral RNA on the nucleus, but not on the
cytoplasmic membranes (Fig. 1). On the other
hand, the 27S and 20S RNAs were associated
largely with the cytoplasmicmembranesatthis
earlystage of virusreplication.Thisobservation
suggests that the sequence of RNA synthesis and the amount of various viral RNAs made may vary with the type of cell membrane
involved. The relationship between the
mem-branes of the nucleus and cytoplasm in JEV
replication deserves furtherinvestigation.
ACKNOWLEDGMENTS
We wish to express our appreciation to Richard Grays and Rodney T. Miller for their excellent technical assistance and to Emilio Weiss for his criticisms and comments in the preparation ofthis manuscript.
Thisinvestigation was supported by the Bureau of Medi-cine and Surgery, Department of the Navy, project no. MR041.05.01-0006B3GJ.
LITERATURE CITED
1. Amako, K., andS.Dales. 1967.Cytopathologyof menin-govirus infection. II. Proliferation of membranous cis-ternae.Virology32:201-215.
2. Baltimore, D. H., H. J. Eggers, R. M. Franklin, and I. Tamm. 1963.Poliovirus-induced RNA polymerase and effects ofvirus-specific inhibitors on its production. Proc. Nat. Acad.Sci. U.S.A.49:843-849.
3. Bellet, A. J.D., R. G.Harris,and F. K.Sanders.1965. Sites ofsynthesisofencephalomyocarditisvirus com-ponents in infected L-cells. J. Gen. Microbiol. 38:299-307.
4. Caliguiri,L.A., and I. Tamm. 1969.Membranous struc-turesassociated with translation and transcriptionof poliovirus RNA.Science166:885-886.
5. Caliguiri,L.A., and I. Tamm. 1970. The role of cytoplas-mic membranes in poliovirus biosynthesis. Virology 42:100-111.
6. Friedman,R.M.,J. G.Levin,P. M.Grimley,and I. K. Berezesky. 1972. Membrane-associated replication complex in arbovirus infection. J. Virol. 10:504-515. 7. Ginzburg-Tietz, Y., E. Kaufmann, and A. Traub. 1964.
Studiesonnuclear ribosomes.2.Transfer of ribosomes from nucleus to cytoplasm in early stages of virus infection.Exp.Cell Res.34:384-395.
8. Grimley, P. M., I. K. Berezesky, and R. M. Friedman. 1968. Cytoplasmic structures associated with an ar-bovirus infection: loci of viral ribonucleic acid synthe-sis.J. Virol. 2:1326-1338.
9. Grimley,P.M., J. G. Levin,I.K.Berezesky, and R. M. Friedman. 1972.Specificmembranous structures asso-ciated with thereplicationofgroup A arboviruses. J. Virol. 10:492-503.
10. Harris, H. 1964. Breakdown of nuclear ribonucleic acid in presence of actinomycin D. Nature (London) 202:1301-1303.
210
on February 25, 2021 by guest
http://iai.asm.org/
11. Hirai, T., and A. Hirai. 1964. Tobacco mosaic virus: cytological evidence ofthesynthesis in the nucleus. Science 145:589-591.
12. Inoue, Y. K., and M. A. Yamada. 1964. Clonal lineof porcine kidney stable cells for assay of Japanese
encephalitis virus. J. Bacteriol.87:1239-1240. 13. Krug, R. M. 1972. Cytoplasmicand nucleoplasmic viral
RNP'sin influenza virus-infected MDCK cells.
Virol-ogy50:103-113.
14. Levin, J. G., and R. M. Friedman. 1971. Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J.Virol. 7:504-514.
15. Lowry, 0. H., N. J. Rosebrough, A. L.Farr, and R. J.
Randall. 1951. Protein measurement with the Folin phenolreagent.J. Biol.Chem. 193:265-275. 16. Martin, E. M., and T. S. Work. 1961.Studiesonprotein
and nucleic acid metabolismof virusinfected
mamma-lian cells. 4. The localization of metabolic changes within subcellular fractionsofKrebs IImouseascites
tumorcells infected withencephalomyocarditis virus. Biochem. J. 81:514-520.
17. Phillips, B. A. 1972. The morphogenesis of poliovirus. Curr. Top. Microbiol. Immunol.58:157-174. 18. Plagemann,P.G. W., and H. E. Swim.1966.Replication
of meningovirus. I. Effect on synthesis of
mac-romoleculesby host cell. J. Bacteriol. 91:2317-2326. 19. Shalla, T. A., and A. Amici. 1967. The distribution of
viral antigen in cells infected with tobacco mosaic virus
asrevealed by electron microscopy. Virology 31:78-91.
20. Takeda, H., M. Yamada, and Y. Aoyama. 1965. Demon-strationof RNAsynthesis caused by Japanese enceph-alitis virus infectioninPS(Y-15) cellswith theaidof chromomycinA,.Japan. J. Med. Sci. Biol. 18:111-120. 21. Takehara, M.1971.Comparativestudiesonnucleicacid
synthesis and virus-induced RNA polymerase activity in mammalian cells infected with certain arbovirus. Arch. Gesamte Virusforsch.34:266-277.
22. Tata, J. R., M. J. Hamilton, and R. D. Cole. 1972. Membrane phospholipids associated with nuclei and chromatin: melting profile, template activity, and stabilityofchromatin. J. Mol. Biol.67:231-246. 23. Zbarsky, I. B. 1972. Nuclearenvelope isolation, p.
167-198. In D. M. Prescott (ed.). Methods in cell physi-ology. vol.5.Academic PressInc..New York.
24. Zebovitz, E., J. K. L. Leong, and S.C. Doughty. 1972.
Japanese encephalitisvirus replication:aprocedure for the selective isolation and characterization of viral RNAspecies.Arch. Gesamte Virusforsch.38:319-327.
211