Saint Louis Encephalitis Viral Ribonucleic Acid Replication Complex

(1)

Saint

Louis

Encephalitis

Viral Ribonucleic Acid

Replication

Complex

ATEEF A. QURESHI AND DENNIS W. TRENT

Department of Microbiology, University ofTexas Medical School at San Antonio, SanAntonio, Texas 78229

Received for publication 26 October 1971

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in

thecytoplasm of infected cells associated with a membranous structure which

sedi-ments with an average value of 250S. The integrity of the complex isdestroyed by

de-tergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid

(EDTA) which differentiates this structure fromcellularpolyribosomes. With

cul-turesinwhich cellular RNA was highly labeled prior to infection, ribosomal RNA

couldnot be demonstrated inthecomplex isolated from EDTA-sucrosegradients.

Single-stranded 43S and the 26S and20S forms of viral RNA were found in the

com-plex.ViralRNApolymeraseactivity in sucrose-gradient fractions sedimented in the

same region as the fractions which contained the pulse-labeled viral RNA. The

polymerase incorporated8H-guanosinetriphosphate into acid-precipitable material

in theabsence ofadded template. It was also found that the replication complex

contains viral-specific proteins.

SynthesisofgroupAarboviral ribonucleic acid

(RNA) andenvelopment of thenucleocapsidare

associated with membranous structures (7, 8, 10,

17,18). Intracellular, complete,group B arbovirus

particles are observed inside membrane-bound

cytoplasmic vacuoles although the role of these

structures in viral macromolecular synthesis and

virion envelopmentis not clear (6, 15, 16).

Cali-guriand Tamm(3, 4)havereportedthat

transla-tionandtranscriptionofpolioviralRNA are

asso-ciated with distinct types of membranes which

canbeseparated by isopycnicsucrose

centrifuga-tion. Inpicornavirus-infected cells, RNA

synthe-sis is initiated in 130S structures found in the

smoothmembrane fraction whichmature toform

the late replication complex that has a

sedimen-tationcoefficientofapproximately250(3, 4).This

mature structurecontainsviral RNApolymerase,

single-stranded RNA, the replicative 20S form,

andthereplicativeintermediate 26S form of viral

RNA(4, 9). Thisstructureis definedasthe

viral-RNAreplication complex

(9).

Cytoplasmic

mem-brane-associated

(MRC)

structuressimilartothe

picornavirusreplication complex arealso thesite

of viral RNAsynthesis in cells infected with the

group A arboviruses (7, 10).

Thiscommunicationdescribes theisolationand

preliminary

description

of a

replication

complex

fromBHK-21/13 cellsinfected with Saint Louis

encephalitis

(SLE)

virus. This structure has an

averagesedimentation coefficient of250,contains

viral RNA polymerase, single-stranded viral

RNA, and the replicative forms of SLE viral

RNA. Viral-specific proteinsappear to be an

in-tegral part of the SLE viralcomplex.

MATERIALS AND METHODS

Virus andcell culture.Pools of SLEvirus and pig

kidneycellmonolayer cultures were prepared

accord-ing to procedures described previously (11, 20). Baby hamsterkidney cells (13) obtained from the American Type Culture Collection were grown in a

lactalbumin medium(20).

Infection procedure. Confluent monolayercultures

of BHK cellswereinfectedwith SLEvirusat a

multi-plicity of 10plaque-forming unitsper cell. After an

absorption periodof 1 hr, warmEagle's medium (5)

with actinomycin D (0.5 ,ug/ml) and 5% calfserum

wasadded, and cultureswereincubated at37C. Cell fractionation. Cultures were pulse-labeled for

15minwith3H-uridine(50,uCi/ml)insingle-label

ex-periments.Indual-labelexperiments,50,ACiof

3H-uri-dine/mland10,ACiof'4C-leucine/mlwereused.After

theradioactive pulse,cells werescraped off theglass

and collected bycentrifugation, andcytoplasmic

ex-tracts were prepared (7). Insomeexperiments,

cyto-plasmicextracts were solubilized withBrij-58 or

so-diumdeoxycholate (DOC; 0.5%for 10 min at0C)

priortosucrosegradient analysis. Extracts were

lay-ered over a

28-mi,

5 to 40% (w/v) sucrose gradient

prepared in eitherreticulocyte standardbuffer (RSB;

reference 22) or ethylenediaminetetraacetic acid (EDTA) buffer (9) andcentrifugedat25,000rev/min

for 120 minat4 CinaBeckman SW-27rotor. Frac-565

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tions collected from the gradients were analyzedfor opticaldensity,trichloroaceticacid-precipitable radio-activity (20), orboth.

Enzyme assay. Viral RNA polymerase activity in cell extracts andgradient fractionswasdeterminedby

themicrotechnique of LevinandFriedman(12).Fifty lambda of the enzyme preparation was incubated at

37 C for 60 min in a total volume of 0.1 ml which contained 0.05 M tris(hydroxymethyl)aminomethane,

pH8.0;0.01 MMgCl2; 0.001 Mdithiothreitol, 0.025jAg

ofactinomycin D, 0.01 M phosphoenol pyruvate;4jg

ofpyruvatekinase, and 10 nmeach ofadenosine tri-phosphate, uridine triphosphate, cytidine

triphos-phate, and 3H-guanosine triphosphate (GTP; 2.9 X

106counts permin).

Analysis ofRNAassociated withcytoplasmic struc-tures. RNA wasextracted frompooled, sucrose gra-dient fractions with sodium dodecyl sulphate-phenol

at 37 C andanalyzedbysucrose ratezonal

centrifuga-tion (20). One-half of each fraction was hydrolyzed

with ribonuclease (20,g/inl), and each sample was

precipitated with 10%ho trichloroacetic acid, collected

on glass fiber filters, and washed several times with

5%70 trichloroacetic acid, and dried, and the radio-activity wasdetermined (20).

Immunoprecipitation of cytoplasmic fractions.

Su-crosegradientfractions(200Sto300S) ofcytoplasmic

extracts of 3H-leucine-labeled SLE viral-infected or

noninfected cellswere pooledanddialyzed overnight

at4 C against 0.02 M phosphate buffer, pH 7.4. An

amount(25,uliters) ofthedialyzedsamplewaslayered

over 25 Muliters of SLE virus antibody-containing

mouseascitic fluid (2) and incubatedat room

temper-ature overnight. A positive test was indicated by a

zoneofprecipitatewhich formedattheinterphaseof

the solutions containing homologous antigen and antibody. Immunological precipitation of 3H-leucine-labeledproteininpooled gradientfractionswas quan-titated by the radioimmune precipitation technique

(19).

Chemicals. Ribonuclease was purchased from

Worthington Biochemical Corporation, Freehold,

N.J., and ribonuclease-free sucrose wasfrom Mann

Research Laboratories, N.Y. Actinomycin D was a

gift of Merck-Sharp and Dohme Research

Labora-tories, Rahway, N.J. 3H-uridine and 3H-guanosine

triphosphate were obtained from Schwarz/Mann,

Orangeburg, N.Y.; 14C- and 3H-leucine were pur-chased from New England Nuclear Corporation,

Boston, Mass.

RESULTS

Cytoplasmic fractions associatedwithviral RNA

synthesis. Cytoplasmic extracts prepared from

noninfected cellsweredivided, and a portion was

incubated with either DOC or Brij-58 before

centrifugation on RSB-sucrose density gradients

(Fig.

1).The A26ogradient profile of cytoplasmic

extracts prepared without detergent treatment

shows materials which sediment with a size

dis-tribution of 300S to 236S, 74S ribosomes, and

lighter material. Brij-58 treatment prior to

gra-dient analysis increased the size distribution of

material in the major A260 peak from 340S to

200S. Sucrose gradients of extracts after DOC

treatmentcontainedrelativelyless material in the

200S to 300Sregion of the gradient.

Cells infectedfor 18 hr were labeledwith

3H-uri-dine for 15 min; cytoplasmic extracts were

pre-pared and treated with either DOC or Brij-58

and centrifuged through preformed RSB-sucrose

gradients (Fig. 2). The gradient profile of

non-detergent-treated cytoplasmic extracts had a

3H-uridine peak that contained approximately

40%o of the total radioactivity and sedimented

with an average value of 300S (Fig. 2A). Three

smallerpeaksofradioactivitywerealso observed

at 250S, 180S, and 140S.Afterincubationof the

extract with ribonuclease, less than

10%,

of the

radioactivity observed in the fast-sedimenting

material of the untreated extract sedimented at

200S to 300S. Incubation of the extracts with

either Brij (Fig. 2B) or DOC (Fig. 2C) prior to

I10

E 236 S

c 0.5

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_ _

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0 5 10 15 20 25 30

FRACTION IUMBER

FIG.1.Effectof sodiuincleoxycholate

anid

Brij-58oln

cytoplasmic extracts fromnolninfcetedBHKcells.

Cy-toplasmic extract was prepared, divided, treated with

either DOC or Brij-58, anid centrifuged through pre-formed reticulocyte stanidard buffer-sucrose

gradienits.

Centtrifugationt was perfbrmed with a SW-40 rotor at

25,000 rev/minifor120 miii at 4 C. Fractiouis (0.5ml)

were collectedanidatnalyzedforoptical

denisity

at 260 mim.Symbols: (0) witreated cytoplasmicextract; (A) Brij-58-treated cytoplasmic extract;(0) deoxycholate-treated extract.

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FRACTION NUMBER

FIG. 2. Effectofsodiumdeoxycholate, Brij-58,and ribonuclease ont thecytoplasmicextractsfrom SaittLouis

enicephalilis virus-infectedcells. At 18hrpostinfection cultures werepulse-labeledwith 3H-uridinte (50 ,uCi/ml)

for 15miii. Cytoplasmicextracts wereprepared, divided, itncubatedwith

deoxycholate

or

Brij-58,

anid

cenztrifuged

throughpreformedreticulocytestandardbuffer-sucrosegradients.Fractiouisof1.0mlwerecollected,anidone-half

of eachfractionwasincubated with10 lAgofribontucleaseat37 Cfor10 miii.Riboniuclease-ireatedand

niontreated

fractions were analyzed for acid-precipitable radioactivity. Total 3H-uridine radioactivity

(@);

ribontuclease-resistant3H-uridineradioactivity (0).A,

nondetergenit

treated;B,

Brij-58

treated; C, deoxycholatetreated.

centrifugation dissociated mostof the 3H-uridine

radioactivity from structures which sediment at

200S to 300S. When the250S 3H-uridine-labeled

peak from infectedcells was isolated from

RSB-sucrose

gradients,

treated with

Brij

or

DOC,

and

recentrifuged,the

gradient

profiles

weresimilarto

those

presented

in

Fig.

2A and

2B,

respectively.

Unlike

polyribosomes

from noninfected

cells,

whichwerestableto

recentrifugation

after

Brij-58

treatment, the faster

sedimenting

3H-uridine-la-beled structures fromvirus-infected cells are

dis-sociatedby the nonionic

detergent.

These results

indicate that SLE viral RNA is associated with

detergent and ribonuclease-sensitive structures

which are distinct from

polyribosomes

observed

inuninfected cells.

Characterization of the

replication complex.

Since the

rapidly

sedimenting

3H-uridine-labeled

structurefromSLEvirus-infectedcellssedimented

in sucrosegradientsina manner similar to

poly-ribosomes,

it was

thought

it

might

contain viral

messenger RNA

(mRNA)

and ribosomes or it

could be a

replication

complex.

Since EDTA is

knownto causedissociationof mRNA and

ribo-somes (9), its effect on the sedimentation

profile

of the

complex

was

investigated.

Cytoplasmic

ex-tracts were

prepared

from cultures labeled with

3H-uridine and '4C-leucine for 30 min at 16 hr

postinfection.

Halfof theextractwas

adjusted

to

0.02 M EDTA and

analyzed

by

sedimentationon

anEDTA-sucrosegradient (Fig. 3A). The control

extract was analyzed by sedimentation on a

RSB-sucrose gradient (Fig. 3B). TheA280 profile

ofthe control

(Fig.

3B) shows two peaks at

ap-proximately250S and 180S,the74S single

ribo-somes,andlighter materials. IntheRSB-sucrose

gradient,most of the3H-uridineradioactivity

re-mained nearthetop ofthe gradientwith asmall

amountoflabeled materialsedimenting inapeak

at2505.TheA28oprofile ofEDTA-treated extracts

(Fig. 3A) did not have a major peak at 180S

(polyribosomes)

but does show a broad peak

sedimenting

in the 200S to 3005 region of the

gradient. Radioactive RNA and protein

cosedi-mented in the EDTA-sucrose gradient in the

peak at 250S.These results differentiate between

structureswhichcontainpulse-labeledviral RNA

andare stablein EDTA(Fig. 3A) andthe

poly-ribosomes which sedimentatapproximately 180S

andaredissociated inthepresenceofEDTA.

To

investigate

whethercellularribosomalRNA

is an integral part of theEDTA-resistant

struc-ture, uninfected BHK cells were incubated in

3H-uridine-containing

mediumfor 24 hrtolabel

cellular ribosomal RNA. After an additional 12

hr in mediumwithoutlabel,the cellswereinfected

for 16hrin the presence of

actinomycin

(0.5

Mg/

ml) andpulse-labeledwith

'4C-leucine

for15 min.

Cytoplasmicextractsprepared fromthe cellswere

divided and sedimented through either

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Fraction Number

FiG. 3.Effectofethylenediaminetetraaceticacid(EDTA)onthereplication complex. 3H-Uridine (50 Ci/ml)

and14C-leucine (10,uCi/ml) wereaddedtocultures 16 hrpostinfection. Incorporation wasstopped after30 min.

(A) partoftheextract wasadjustedto0.02 MEDTAand sedimentedinanEDTA-sucrosegradient. (B) control

wassedimentedin an reticulocyte standardbuffer-sucrose gradient. Symbols: (0) acid-precipitable 3H-uridine

radioactivity; (0) acid-precipitable 14C-leucinieradioactivity; (-) A280.

sucrose orRSB-sucrose

gradients.

Fig.

4Ashows

thatuponEDTAtreatmentmostofthe3H-uridine

in RNA from prelabeled cells sediments at the

top of the

gradient

at

approximately

745 with a

coincident

peak

of

3H-uridine

and '4C-leucine

radioactivity

sedimenting

at

approximately

250S.

In contrasttothis, RSB-sucrose

gradients

of

ex-tractsfromprelabeledcells

(Fig.

4B)

contained a

large prominent peak of 3H-uridine and

'4C-leu-cine radioactivity which sedimented at 180S to

250S.

Since a small percentage of 3H-uridine in the

EDTA-sucrose gradient sedimented inthe

heavy

regionof the gradient (Fig. 4A), fractions inthe

200Sto250S region of the

gradient

werepooled

and resedimentedin EDTA

(Fig.

4C).

Resedimen-tation ofthe 250S peak in EDTA shifted more

than 90% of the 3H-uridine counts in these

pooled fractionstothe topof thegradient.These

data indicate that prelabeled cellular ribosomal

RNAispartially dissociatedto structures smaller

than250SbyEDTAtreatment andthat some of

theremainingRNAwhichsedimentsat250Swill

partially dissociatetosmallerstructuresif treated

againwith EDTA. Theproteinin the250S peak

whichwaslabeledwith 14C-leucineduring the

in-fectionwas notdissociated from the complex by

this repeated EDTA treatment. These data

sug-gestthatmostofthecellularRNAfrom the

pre-labeledcellswhich sedimentswith the 250S

struc-tureisloosely bound.

Todetermine the type of RNA left in the 250S

region of thegradient from prelabeled cells after

the second centrifugation in EDTA, fractions 6

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10 20 30 10 20 30 10 20 30

Fraction Number

FIG.4.Absenceofribosomes inthe replication complex. BHK cells were prelabeled with 50 ,uCi of3H-uridine

per mlfor 24 hr and then 12 hr in the absence of the isotope. The cells were infected for 16 hrinthepresence

ofactinomycinandpulse-labeledwith14C-leucine(10,uCi/ml)for 15 min,andcytoplasmic extractswereprepared. (A) partofthe extractwasadjusted to 0.02 Methylenediaminetetraacetic acid(EDTA)and sedimentedthrough anED TA-sucrose gradient. (B) control was sedimented through a reticulocyte standard buffer-sucrose gradient.

(C)fractions5to 15of the EDTA gradient indicated by the arrow in Fig. 4A were pooled, dilutedwithone-half

volume of ED TA buffer,anidanalyzed in an EDTA-sucrosegradient. Symbols: (-) trichloroacetic acid-precipitable 3H-uridineradioactivity; (0) trichloroacetic acid-precipitable14C-leucine radioactivity; (-) A280.

to 18 of the gradient shown in Fig. 4C were

pooled,and the RNAwasextracted andanalyzed

in sucrose gradients. Aheterogenous mixture of

ribonuclease-sensitive RNA species were found

with about 22% ribosomal, 3% single-stranded

viral43Sand theremaindersmall RNA species.

The RNA from the prelabeled 2505 peak

sedi-mented only in RSB(Fig.4B)was90%ribosomal

RNA.

Table 1 shows the percentage of 3H-uridine

counts remaining in the 250Sregion ofthe

gra-dient following centrifugationin RSB and

recen-trifugationin EDTA.Followingthe first

sedimen-tation in RSB, 82% of the RNA from the

pre-labeled cells was observed to sediment in the

200S to 3005 region ascompared to 15% inthe

extractsedimentedthroughEDTA.

Recentrifuga-tion of this 250S peak from prelabeled cells in

EDTA dissociated about 90% ofthe remaining

RNAfromthe complex to sediment nearthe top

of the gradient (Fig.

4).

The 250S

peak

from

pulse-labeled cells containedapproximately 15%

of thetotal RNA in EDTAgradientswhich did

not dissociate from the complex after isolation

andrecentrifugation.

To characterize thespecies of RNAwhich are

contained in thestructureswhich sediment in the

250S regionofthe gradient frominfected

pulse-labeled cells, cultures were infected and were

pulsed with 3H-uridine for 30 minat 16hr

post-infection, and the 250S peak was isolated from

EDTA gradients. The RNA was extracted and

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analyzed on sucrose gradients (Fig. 5). Three

species of viral RNA areassociatedwiththe 250S

cytoplasmic structure from the infected cells. The

largest species has a sedimentation coefficient of

43, is ribonucleasesensitive,and isassumedto be

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newly synthesized single-stranded progeny type

TABLE 1. Absence of ribosomes in the replication complexa

Per centRNAremaining in the 250Sregion of thegradient Type ofgradient

Pulse-labeled Prelabeledb infected infected

RSB 15 82

1st EDTA 17 17

2nd EDTA 12 0.01

a RSB, reticulocyte standard

ethylenediaminetetraacetic acid.

bData taken from

Fig.

4.

25

loo 43s

28 S

E 75;1

50 26s 2

50.

C)

25.

PO

I I5 Ib

\

/1t\

+

buffer; EDTA,

FRACTION

NUMBER

FIG. 5.Sucrosegradientanalysisof RNAextracted from viral replication complex. Cultures infected for 16

hr in the presentce of

actinomycint

were pulsed with

3H-uridine for 30 miii; extracts were prepared and

centrifuged through ethylenediaminetetraacetic

acid-sucrosegradients.TheRNAinthe250S peak was then

extracted with sodium dodecylsulfate-phenol and an-alyzedon sucrose gradients. Trichloroacetic acid-pre-cipitable counts before (0)andafter(0) ribonuclease treatment. The designation 28S indicates the optical density peak of ribosomal RNA.

RNA. The 26S

partially

ribonuclease-resistant

and 205 ribonuclease-resistant forms of viral

RNAwerealso present

(20).

These data lead to the conclusion that viral

RNA is contained in the 250S structure as a

tightly

bound

integral

part of the complex to

which small amounts ofribosomalRNA species

maybelooselyassociated.

RNA polymerase. Since the fast-sedimenting

material in the cytoplasmic extract contained the

replicativeforms of viralRNA,experimentswere

donetodetermineifviral RNApolymerase

activ-ity was present in the 250S particulate fraction.

Cytoplasmicextracts werepreparedfrom cultures

infected for 18 hr and analyzed on sucrose

gra-dients. Polymerase activity in gradient fractions

was determined by measuring the incorporation

of 3H-GTP into acid-precipitable material. As

shown inFig. 6, fractions8to 13 containedmost

of the enzyme activity which sedimented in the

sameregionofthegradientaspulse-labeled RNA

(Fig. 3B). Treatment of extracts with DOC or

Brij-58 either prior to or after gradient analysis

destroyedall detectable enzymeactivity.

SLE viralpolymerase hasproperties similar to

those reported for the group A arboviruses (14,

18) (Table 2). The enzyme required magnesium

ions and all four nucleotide triphosphates and was

inhibited by manganese ions and ribonuclease.

Incorporation of 3H-GTP into acid-precipitable

material was linearduring the first hour of

incu--- 8

74s A

100

-O .~i

t6 1,

C,

-4 C. 3

F,o--tio, N,rmbe,

FIG. 6. Sedimentation pattern of RNA polymerase

in cytoplasmic extract from cells infected with SLE virus. BHK cells wereinfected for 18 hr; cytoplasmic

extractwasprepared and centrifuged through an ethyl-enediaminetetraacetic acid-sucrose gradient. Fractions (1.0 ml) were collected and analyzed for RNA poly-meraseactivity.

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TABLE 2. Effect ofMII2+, Mg2+,ntcleotides,

actinto-mycini D, ribolnuclease, and adeniosi,ie

triphos-phate-genteratinig system olt Sainlt Loulis

enl-cephalitis viralpolymerase

Total Reactionsystema radio-activityb

Complete assay... 1,572

Complete assay without

Mg2... 315

ATP . 173

CTP 250

UTP . 187

ATP, CTP, UTP 73

Phosphoenol pyruvate and

phospho-enol pyruvate kinase 817

Actinomycin D 1,436

Complete assay with

Ribonuclease (0.5 jAg) 506

Mn'+... ... 346

Uninfected cells (complete assay) 184

aCTP, cytidine triphosphate; UTP, uridine triphosphate.

bCounts per minute of

3H-guanosine

ttiphos-phate incorporated into acid-precipitable ma-terial.

bation and then declined rapidly. Cytoplasmic

extracts of uninfected BHK cells did not

incor-porate a significant amount of 3H-GTP into

acid-precipitable material in the presence of

actinomycinD.

Viral proteins in the replication complex.

Im-munoprecipitationtests wereperformedto

deter-mineiftheprotein associated with thereplication

complex wasviralspecific. Fractions inthe 250S

region of the gradient shown in Fig. 3A were

pooledand reacted withSLEviral-hyperimmune

ascitic fluid. A visible zone of precipitate

devel-oped at the

interphase

between the SLE viral

antibodyandfractionswhichcontained the

repli-cationcomplex.Noprecipitatewasobservedafter

incubation of2505 gradient fractions from

non-infected cellswithviral antiserum or when normal

ascitic fluid was used. Most (92%) of the

radio-activity in those fractions which contained the

replication complex was precipitated with SLE

viral antibody, in comparison to less that 10%

fromcytoplasmic fractions of noninfectedcellsor

when normal, mouse ascitic fluid was used. The

replication complex therefore does contain

viral-specific proteins. Whether these proteins are

structural, nonstructural, or

both,

is not known

(21).

Kinetics oflabelingthereplication complex. The

experimentsdescribed above have established the

existence of a cytoplasmic replication complex

(CRC) in SLE virus-infected cells. To determine

the time in the growth cycle when this complex

was most active in RNA synthesis, cultures

in-fected for 12 hr and 18 hr were doubly labeled

with3H-uridine and '4C-leucine for 15 min.

Cyto-plasmic extracts from these cells were analyzed on

sucrose-EDTA gradients (Fig. 7). It is observed

thatfractions which containmaximal protein and

RNA (trichloroacetic acid-precipitable

radioac-tivity) arecoincident in the gradient. The amount

of "4C-leucine counts in thefractions containing

the CRC from 12-hr(Fig. 7A) and 18-hrinfected

cells (Fig. 7B) is similar, whereas the amount of

3H-uridine radioactivity in the 2505 region from

the 18-hr cultures had increased approximately

twofold. The CRC in SLEviral-infected cells

ap-pears to contain either a constant amount of

structuralprotein, enzymatic protein, or both, or

at 12 hrpostinfectionthe synthesis and release of

protein in the complex has reached a constant

rate. These experiments do not distinguish

be-tween thesepossibilities.However, since the CRC

does not appear to contain polyribosomes and

therefore isprobably notinvolvedinprotein

syn-thesis, we feel that these data probably reflect the

CRCcomposition. The increasedincorporation of

3H-uridine into viral RNA in the replication complex during the viral growth cycle is

concomi-tantwith the increase in totalcell-associated viral

RNA synthesis (20).

DISCUSSION

Pulse-labeled SLE viral RNA is found in a

2505 cytoplasmic structure which contains viral

RNA polymerase activity and the nascent and

replicative forms of viral RNA. These results

sug-gest that this structure is a replicationcomplex,

i.e., the site of SLE viral RNA synthesis. The

replicationcomplexis first detected 6 hr after

in-fection (unpublisheddata) and is most active in

RNA synthesis during the rapid phase of viral

growth when the rate of viral RNA synthesis is

maximal(20).

Input Semliki Forest viral RNA bindsto

mem-braneousstructureswhich have many similarities

tothe SLE viral MRCstructures (8, 17). The

ob-servationthatcycloheximide inhibits the binding

ofSindbis viral RNAtothe MRCstructuresmay

explain our previous observation that

cyclohexi-mide treatment prior to the end of the latent

period inhibits SLE virusgrowth (21).Incontrast

to the polioviral replication complex (9), the

structure and functional

integrity

of the MRC

structures from the Sindbis (17) and SLE viral

CRCare destroyed by

detergent

treatment. This

wouldsuggestthat the arboviralreplication

com-plex may be composed of subunits which are

membrane associated. Dissociation of these

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0 5 15 25 35 5 15 2 5 35

FRACTION NUMBER

FIG. 7. Sucrose gradient analysis ofthe replicalion complex pulse-labeled early andlate during viral RNA

synthesis.BHKcellsinfectedfor12hr and 18 hrweresimultaneously pulse-labeledwith3H-uridineand"4C-leucine

for15min; cytoplasmicextracts werepreparedandcentrifuged through ethylenediaminetetraaceticacid-sucrose

gradients. Symbols:(0) 3H-uridine trichloroacetic acid-precipitableradioactivity; (0) 14C-leucinetrichloroacetic

acid-precipitable radioactivity.A, 12 hrpostinfection;B, 18 hrpostinfection.

subunits

during the

isolation

procedure may

ex-plain

the failure of arboviral RNA

polymerase

preparations to

synthesize

progeny-type,

single-stranded RNA (14).

Grimley

etal.

(10)

have

de-scribed adistinct type ofcytoplasmic vacuole in

Sindbis virus infections

which

they

believetobe

the site of viral RNA

synthesis.

Many smaller

vacuoles with similar features are found

in

the

perinuclear region of SLE virus-infected cells,

however, their rolein viral RNAsynthesis isnot

now clear(15;unpublisheddata).

In contrast to picornaviral polyribosomes

which are stabletodetergent treatment and can

readily be isolated(9),repeatedattempts toisolate

viral polyribosomesfromSLEviral-infectedcells

using many techniques have

failed

(unpublished

data).

TheSLE viral CRCdoesnot appear tobe

associated with viral protein synthesis or to

con-tain ribosomes. The cellular site of SLE viral

proteinsynthesis isnot nowknown. Thepresence

of viral

antigen(s)

intheCRCmay beexplained

by theincorporation of viral protein(s) into the

membranes from whichthe complex develops or

associates. TheSindbis viral envelope isacquired

bybudding of the

nucleocapsid

through

cytoplas-mic or internal membranes (10). The envelope

contains a single polypeptide which gives the

virion

hemagglutinating, complement-fixing

and

neutralizing antibody-blocking

activities (1). Viral

antigen in the SLE viral CRC may reflect the

presence of a viral-modified membrane as a

structuralpartofthisstructure. Antigens inthe

CRCmaybe identicalto theenvelope

polypep-tides of the complete SLE virion (21). Further

experimentsareneededtocharacterizethe

mem-branes associated with the replication of SLE

viral RNA,stiuctural and nonstructuralproteins,

and theenvelopmentprocess.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service research grantAI-09397 from the National Institute of Allergy andInfectious Diseases.

I

on November 10, 2019 by guest

http://jvi.asm.org/

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