Control of protein synthesis in Semliki forest virus-infected cells.

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Control of Protein

Synthesis

in

Semliki Forest Virus-Infected

Cells

BAT-EL LACHMIl* AND LEEVI KAARIAINEN

DepartmentofVirology,Universityof Helsinki, SF-00290Helsinki29, Finland Receivedfor publication 3 August 1976

Protein synthesis in Semliki forestvirus-infected chicken embryo cells was

studied by labeling them with [35S]methionine for short periods at different

timesafter infection,with orwithout synchronization ofproteinsynthesisbythe

hypertonic block technique. The rate ofhost-cell protein synthesis declined

almost linearly in inverse correlation to the increase in the amount of

virus-specific RNA. At5.5hpostinfection, the host-cell proteinsynthesiswasreduced

byabout 70%. The viralstructuralproteins weredetectablewithcertaintyat3.5

h postinfection, and their rate of synthesis increased linearly parallel to the

amount of their messenger, the 26S RNA. This suggests that the rate of

synthesisof the structuralproteins is controlledatthe level oftranscription.The

rate of synthesis of the virus-specific nonstructural proteinsattained its

maxi-mum between 3 and 4 h postinfection and declined thereafter, whereas the

amountof theirmessenger, the42SRNA, continuedtoincreaselinearlyinthe

cells.Thus,the messengeractivityofthe42S RNA is reducedinthelatephaseof

infectioncompared with its activity inthe earlyphase.

The mode of expression ofthe genetic

infor-mation ofSemliki forest virus 42S RNA

(molec-ularweight, 4.0 x 106to 4.5 x 106) (19, 26) has

recently been elucidated to alarge extent.The

fourstructuralproteins(capsidprotein and the

glycoproteins El, E2, and E3) are translated as

apolyprotein with a molecularweightofabout

130,000 (16, 17). The messenger for the

struc-turalpolyproteinis theintracellular 26S RNA

(molecular weight, 1.6 x 106) (26), which is a

replicaof the 3' third of the 42S RNA (38). The

translation startsfromoneinitiation site ashas

beenshown by several investigators (5, 7, 9).

Four nonstructural proteins with molecualr

weights of70,000 (ns7O), 86,000 (ns86), 72,000

(ns72),and60,000(ns6O)wererecentlyshownto

be synthesized sequentially when a

tempera-ture-sensitive mutant (ts-1) of Semliki forest

virus was studied (18) by the hypertonicblock

technique (6, 24, 25). The four nonstructural

proteins were apparently synthesized as a giant

polyprotein with a molecular weight of about

290,000, which is presumably cleaved to give

two short-lived intermediates with molecular

weights of 155,000 (nsl55) and 135,000 (ns135)

(18). The messenger for the nonstructural

pro-teins is probably the viral 42S RNA genome,

whichinvitrodirects thesynthesisofproducts

identical to the nonstructural proteins isolated

' Present address:Department ofVirology,Israel Insti-tuteforBiologicalResearch, Tel-AvivUniversityMedical School, P.O.B. 19,Ness-Ziona,Israel.

from ts-1-infected cells (N. Glanville,B.Lachmi,

A. E. Smith, and L.Kaariainen, submitted for

publication). Support for the idea that the

non-structural proteins are made as a polyprotein

has also been obtained from in vitro studies, in

which the 42S RNA-directed product was

shown toyield only oneformyl-[35S]methionine

tryptic and pronase peptide, suggesting that there is only one active initiation site for the nonstructural proteins in this RNA (9).

Here we have studied the synthesis of the

structural and nonstructural proteins in

Sem-liki forest virus wild-type-infected cells. The

rate of synthesis ofthe nonstructural proteins

was maximal early in infection and declined

thereafter, despite the continuous increase in

the amount of the 42S RNA. Bycontrast, the

rate of synthesis of the structural proteins in

thecells increasedparalleltothe amountof 26S

RNA showing that the synthesis of structural

andnonstructuralproteins is controlledby

dif-ferent mechanisms.

MATERIALS AND METHODS

Virus and cells. Semliki forest virus prototype strain (13)and a ts-1 mutant (15, 16) were used in these studies. BHK-21 cells and secondary special

pathogen-freechicken embryo fibroblasts were cul-tivatedasbefore (16). All experiments werecarried outat39°C,therestrictive temperature,in the pres-enceof1 ugofactinomycin D per ml.

Labeling of virus-specific proteins. Confluent monolayers ofsecondary chickenembryofibroblasts 142

on November 10, 2019 by guest

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ALPHAVIRUS-INFECTED

wereinfectedat amultiplicityof 50 or 500 PFU per cell andincubated at390C. At the times indicated,

the cellswerepulsedwith[35S]methionine,50 or200

jCi

per plate (210 to 250 Ci/mmol, Amersham/ Searle Corp.) in a methionine-free medium. The pulses were followed by chases in the presence of 10-fold the normal concentration of unlabeled methio-nine. The whole cells were taken into hot 2% sodium dodecyl sulfate (SDS) as described before (16, 17). In some experiments the cells were incubated in the presence of 335 mM NaCl for 30 or 40 min before the pulse was given in isotonic medium as described

(18). Protein determinations were carried out

ac-cording to Lowry et al. (20) using bovine serum albumin as standard.

Polyacrylamide gel electrophoresis. Polyacryl-amide gel electrophoresis was carried out by the

discontinuoussystemdescribedby Neville (23) and modified as before (17). Electrophoresiswas carried out inslabgelsat 10 mAfor 6 to 7 h. Thegelswere dried and autoradiographed. In some experiments after electrophoresis the gels were impregnated

withPPO (2,5-diphenyloxazole) andthen dried and

fluorographed (2). Forquantitation, theindividual lanes, taken from the slab, were cut into 1-mm slices, solubilized in NCS (Nuclear Chicago Solubi-lizer) and counted in a toluene-based scintillation fluid. The quantitations of the gels were done

ac-cordingtothe methoddescribedbyMcAllister and

Wagner (21). This method enabled us to determine the percentage ofreductioninhost-cellprotein accu-mulationand the accumulation of virus-specific pro-teins.

The region of each gel that does not contain knownvirus-specific proteins (fractions 5 to 15) was chosen to normalize activity. The normalization ra-tio (N), which indicates thedegree ofreduction of host-cellproteinsynthesis,wasdeterminedby divid-ing the sum ofradioactivityinfractions 5 to 15 from virus-infectedcells(I)bythe sum ofradioactivityin the same region from mock-infected cells (U), i.e., N

=I/U.

The virus-specific protein accumulation in each infected cell gel fraction (V) was determinedbythe equationi-NU,where iwastheradioactivityinan infectedgelfraction and U was theradioactivityin the corresponding mock-infected gel fraction, and where N is thereductioninhost-cell protein

synthe-sisand NU is the host-cellbackgroundestimated for theinfectedcellgelfraction.

Labeling of viral RNA. Confluent monolayers

wereinfected at amultiplicityof 500 PFUand incu-bated at39°C.Atthe end of theadsorptionperiod,10

,ACi

perplateof[3H]uridine(28Ci/mmol)wasadded toeachplate, and the incubation was continued. At 1-h intervals, from 1.5 to 5.5 h postinfection, one plate was taken into 2% SDS, and the extracts were

analyzed in15to30% (wt/wt) sucrose gradients as described before (15).

RESULTS

Identification of Semliki forest

virus-in-duced proteins. The structural and

nonstruc-tural proteins synthesized in chicken cells

in-fected withour ts-1 mutant at39°C are shown

inFig. 1. As shown, the viral proteinsynthesis

recovered more quickly from hypertonic block

ofinitiation than did the host-cell protein

syn-thesis (6, 18, 24). Wetriedtoreduce the

resid-ualhost-cellprotein synthesistotheminimum

in infected cellsby incubating them in

hyper-tonicmedium (335 mMNaCl) for 40 min. When

the hypertonic medium is replaced by an

iso-tonic one, synchronous initiation follows (6).

Therefore, infected cellswerelabeled

immedi-155

135

86

72

U.701

p62

mw

C

-El

-C

A

B

FIG. 1. Autoradiograph of7.5% polyacrylamide

slabgel of cellular extractsfromts-1-infected chicken

embryo cellsfollowing synchronizationof initiation byahigh-saltblock. Infected cells were incubated at 39°C for4h 20min, treated with high salt (335 mM NaCl) for 40 min, and then pulsed for 15 min with [35S]methionine (100 uCi per plate) upon restoration

ofsalt concentrations andchased for 15 min (A) or 60 min(B)inthepresence of cold methionine. The slab gelwas impregnated with PPO and then dried and

fluorographedfor1 week. C, El, and p62 are

struc-tural proteins and 155 (ns155), 135 (ns135), 86 (ns86), and 70(ns7O) are nonstructural proteins.

VOL. 22, 1977

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ately with [35S]methionine for 15 min and

chasedinthepresence of excess unlabeled

me-thionine for 15 min (lane A) and 60 min (lane

B). Thefastestmigrating band in the

autoradi-ogramofadiscontinuous SDS slab gel was the

viral capsid protein (C) followed by the

enve-lope protein (El) and the precursor of enveenve-lope

proteins E2and E3 (p62). Thebands migrating

more slowly than p62 were the nonstructural

proteins with apparent molecular weights of

70,000, 72,000 (previously determined 78,000

from itsmigrationin11%gels[16]), and 86,000.

These proteins weredesignated ns7O, ns72, and

ns86 according to the recommendation of an

international group of virologists (1).Inlane A,

which represents the 15-min chase, two

addi-tional bands with apparent molecular weights

of 155,000 (nsl55) and 135,000 (nsl35) can be

seen. These proteins were scarcely detectable

after the 60-min chase suggesting that they

were precursors forthe nonstructural proteins.

The fourth nonstructural protein, ns6O, could

notbe separated from p62 using the usual

poly-acrylamide gel electrophoresis system. It has

only been demonstratedby C-terminal labeling

aftertreatmentwithpactamycin (18).

We have recently shown by tryptic peptide

analysis that ns155 and ns135aredifferent

pro-teins, the former being the precursor for ns7O

and ns86 and the latter for ns72 and probably

ns6O (Glanville et al., submitted for

publica-tion).

Nonstructural proteins in Semliki forest

vi-rus wild-type-infected cells. The function of

the more stable nonstructural proteins is not

yet known. SinceSindbis virus RNA-

tempera-ture-sensitive mutants fall into four

comple-mentation groups (3, 33), one would expect at

least some of the nonstructural proteins to be

involved in the replication of the viral RNA.

Since RNA synthesis is an earlyevent inthe

infectious cycle, the presence of the

nonstruc-tural proteins in wild-type-infected cells was

studied mainly during the early phase of the

growthcycle.

Wild-type-infected cells were labeled at

dif-ferenttimes postinfection both with and

with-out hypertonic salt treatment. The

autoradio-.

6

-,. ... S-*

86

L.-.r

I \

{-1

PfnE,#

; it _W

;_I0,o

*rC_3/1 >

__awL

"Q.",

..wj

4^EI

FIG. 2. Autoradiographof 7.5%discontinuousSDS-polyacrylamide gel of cellular extracts from Semliki forestviruswild-type-infectedandmock-infected cells. Infected chicken embryo cellswereincubatedat39°C

andat2.5h(A),3.5h(C), 4.5 h (D),and5.5h(E); one platewas treatedwithhigh salt (335mMNaCl) for 30min.The cellswerepulsedimmediately with[35S]methionineinisotonicmediumfor20minandchasedfor

5 min. Mock-infected cells (B) were not treated with high salt but were pulsed and chased similarlyat 5 h postinfection. Each sample applied to thegel contained about 180,000 cpm. C and El are the structural proteins. p62 is aprecursorofenvelope proteins E2 andE3. 72, 86, 135, and155 are the nonstructural proteins.

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graph ofa polyacrylamide slab gel in Fig. 2

shows the [35S]methionine-labeled proteins of

Semliki forest virus at 2.5, 3.5, 4.5, and 5.5h

postinfection. The infected cells were treated

with the hypertonicmedium for30 minprior to

the 20-minpulse given inisotonicmedium

fol-lowed bya5-minchase. Inadditionto theabove

describedstructuralproteins,theenvelope

pro-tein E2 is seen between p62 and El (Fig. 3).

The nonstructuralproteinsns155 and ns135are

clearly identifiable,as are ns86, Ms72,and ns7O.

Similar labeling carried out with cells not

ex-posed to the high salttreatment is shown in

Fig.3.The presenceofthe abovedescribed

non-structural proteins among host-cell proteins

canbeseenclearly at3.5hpostinfection (lane

C) with the exception of ns7O, which is not

properlyresolved from ns72.

Individual lanes of the dried slab gels were

cut into 1-mm slices and then solubilized and

counted in liquid scintillator (Fig. 4). To

esti-mate the proportions of viral structural and

nonstructuralproteins, the difference analysis

method of McAllister and Wagner was used

(21).Aregionofthe gel fromvirus-infected and

mock-infected cells (fractions5 to 15 in Fig. 4)

was chosen for evaluation of the reduction in

host-cell-specific protein synthesis. The total

radioactivityinthis region ofgels frominfected

cells was divided by the radioactivity in the

same region ofmock-infected cells. This

nor-malizationratioexpresses thedegreeof

inhibi-tionofhost-cellproteinsynthesis(Fig. 5A).The

multiplicationof theradioactivityin eachof the

mock-infected gel fractions by this ratio gave

the value ofresidual host-cell protein

synthe-sis. This value was then subtracted from the

radioactivity in the corresponding fractions of

thevirus-infected gelsto givethevirus-specific

radioactivity. The amount ofvirus-specific

ra-dioactivity atthe positions ofcapsid, envelope

proteins El, E2, and p62 were summed up to

yield the total radioactivity of structural

pro-teins synthesizedatdifferent timesafter

infec-155

^-

135 _72

l

#*-At

p

Q2

sa

E2

El

C-,l

A

B

C

D

E

FIG. 3. Autoradiograph of7.5%discontinuousSDS-polyacrylamide gel ofcellularextractsfromSemliki

forestvirus-infectedandmock-infectedcells.Infectedchickenembryocellswereincubatedat39°Candat2h 10 min(A),3 h10min(C),4h 10 min(D), and5h 10 min(E)postinfection; theywerepulsed for20min with 50,Ci of[35S]methionineperplateandthen chasedforanadditional5min.Mock-infectedcells(B)were

pulsedandchasedsimilarlyat5h10minpostinfection. The slabgel wasdried andexposed for2 days.

Symbolsand amountsof radioactivityare asfor Fig.2. The mainhostproteinsaremarked withH. 40maw

H

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94-x

U3-I

2

10 20 30 40 50 6 70 80 90 100

FRACTION NUMBER

8 B 155135 66 7n 62 E2 El C

7-

6-

5-o-

A

U3

2-10 20 30 40 50 60 70 80 90 100

FRACTION NUMBER

FIG. 4. Quantitationofpolyacrylamide gels representingwild-typevirus-infectedcellsat3.5h(A)and5.5

h(B)postinfection, representinglanesC and EinFig. 3, respectively.Theprofile ofthequantitation of

mock-infectedcells (lane B inFig.3) isdrawnfor reference. Thearrowsshow thepositionsofnonstructural155

(nsl155),135(ns135),86(ns86), 72(ns72),and structuralproteins El,E2and C.Symbols: 0,virusinfected;

mock-infected.

tion. The result isexpressed asthe percentage

of totalradioactivity recovered from thegelsin

Fig. 5C.

Similarly, thevirus-specificradioactivitiesat

the positions of ns155, ns135, ns86, and ns72

plus ns7O were summed upto yield the total

amount of nonstructural proteins synthesized

duringthe 20-min pulse. In this quantitation

the fourth nonstructural protein ns6O, which cannot be distinguished from p62 by the gel

electrophoresis method used, was included in

the structural proteins. Thus the amount of

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z

rj60-20

2 4 2 4 2 4

HOURS

FIG. 5. Synthesis rate ofhost-cell proteins (A), virus-specific nonstructuralproteins (B), and struc-turalproteins (C) compared with theamountof42S RNA (B) and 26S RNA (C)atdifferent timesafter infection. Lanes of the slabgelshowninFig.3were

cut into 1-mm slices and then solubilized and

counted in a liquid scintillator. The radioactivity

profile from mock-infected cells, showninFig. 4,was

usedtocalculate the degree of inhibition of host-cell protein synthesis and for the estimation of virus-specific nonstructural and structural proteins

ac-cording to the difference analysis method of Mc-Allister and Wagner(21).The radioactivities ofhost-cellproteins and viral nonstructural and structural proteinsarepresented aspercentageof the total

ra-dioactivity recovered from the gels. The cumulative amountof 42S RNA (B) and 26S RNA (C)at differ-enttimesofinfectionareshown for comparison.

La-beling of cells with[3Hiuridine was carriedoutas

describedinthelegendtoFig.6.Symbols:0,rateof

proteinsynthesis by the difference analysis method;

A, residual host protein synthesis determined by

dividingthesumofradioactivityinfractions5 to 15 (see Fig. 4) of infectedcells byrespectivesumfrom

uninfected cells x 100; 0, percent radioactivity in

42SRNA and in 26S RNA oftotal at 5.5 h post-infection.

radioactivity inthenonstructural proteins

rep-resentsanunderestimate(Fig. 5B). Therestof

theradioactivity inthe gelswastaken ashost

proteins givinganother estimate for the

resid-ual hostprotein synthesis (Fig. 5A).

There isaprogressive decreaseinthe rate of

host-cellprotein synthesis and asimultaneous

increase in therateofsynthesisofthe

nonstruc-turalproteins. Themaximum rate ofsynthesis

of the nonstructuralproteinsis reached at 3.5 h

postinfection, ata timewhen host-cell protein

synthesis is inhibited only by about 20%. At

thistime the synthesisrates ofstructural and

nonstructuralproteinsarealmostequal. At 5.5

h postinfection, the synthesis rate of the

non-structural proteins is about half of the

maxi-mum. The difference analysis revealed that

1.4%of the totalradioactivitywasin

nonstruc-tural proteins at 2.5 h postinfection, whereas

nostructural proteinscould be detected atthis

time.

Amount ofvirus-specificRNAs. Cumulative

labeling of wild-type-infected cells with

[3H]-uridine starting from 1 h postinfection was

carried out in the presence of actinomycin D.

Part of the cultures were removed at hourly

intervals, and the RNAs were analyzed, after

treatment with SDS, by sucrose gradient

cen-trifugation as shown in Fig. 6. The

radioactiv-ity in the viral RNAs was taken to represent

the total amount of these RNAs in the cell (35).

The main RNA species are the 42S RNA

ge-nomeand26S RNA, whichweresynthesizedin

a molar ratio of close to 1 at all times after

infection. The increase in the radioactivity in both RNA species is almost linear beginning from 2.5 h postinfection (Fig. 5). The increase in

the rate ofsynthesis of the structural proteins

correlates well with the increase in the amount

of the 26S RNA; however, there does not seem

tobe asimple correlation between the rate of

synthesis of the nonstructural proteins andthe

amountof the42S RNA, which is regarded as

themessengerofthese proteins (5, 8, 9).

15 42S 26S

10

C,,

x

10 20 30

FRACTION NUMBER

FIG. 6. Sucrosegradient analysis ofRNAsfrom

Semlikiforestvirus-infectedcells.Secondarychicken

embryo fibroblasts were infected with 500PFU per cell and maintained in minimal essential medium

supplemented with 0.2% bovineserum albumin in the presence of1 Mgof actinomycin Dper ml. The cellswerelabeled with[3H]uridine(101iCiperplate) from1.0 to5.5hpostinfection. Thewhole cellswere dissolvedin 2%SDSin a buffer containing 0.15 M

NaCl, 0.001 MEDTA, and 0.01 MTris(TSE; pH 7.4). Theextract wasanalyzedin 15 to 30%(wtlwt) sucrosemadeinTSEcontaining0.1%SDS.

Centrif-ugation was for12 h at 24,000 rpm in an SW27

Spincorotor at230C.

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148

LACHMI AND KAARIAINEN

DISCUSSION

The translation of Semliki forest virus-coded

proteins consists of two different processes. (i)

The structural proteins are translated concep-tually as a polyprotein. Due to the nascent

polysomal cleavage ofcapsid protein and the

rapid cleavage between envelope proteins El

and p62 (the precursor of E2 and E3), these

three proteins are the earliest detectable

prod-ucts of translation (6, 30). (ii) The

nonstruc-tural proteins areprobably also translated as a

polyprotein, which should have a molecular

weight to 300,000 (9, 18). Such a protein has not been found, which may indicate that a nascent

cleavage, similar tothatof the capsidprotein,

takes place, giving rise to the short-lived

pre-cursorsnsl55 and nsl35. The former gives rise

to two morestablepolypeptides, ns7Oandns86,

and the latter gives rise to polypeptides ns72

and ns6O (18; Glanville et al., submitted for

publication).

These nonstructural proteins have thus far

been demonstrated only in cells infected with

our temperature-sensitive mutant ts-1 (15-17)

where they accumulate in easily detectable

quantities (11, 12). Inthisstudy we have

dem-onstrated the presence of the nonstructural

pro-teins also inthe wild-type-infectedcells. Their

synthesis was followed during the early hours

of infection and reached its maximum rate at

3.5 h postinfection. At this time, they were

synthesized at a rate similar to that of the

structural proteins (Fig. 5). Estimates at 2.5 h

postinfection, although less reliable because of

the ongoing host-cell protein synthesis, would

suggest that more nonstructural than

struc-tural proteins were made at this time. All our

attempts todemonstratethesynthesis of either

nonstructural or structural proteins earlier

than 2.5 h postinfection have failed. It seems

that at least 10% of the amount of viral RNA

foundat 5.5 hpostinfection mustbepresented

before the proteins become detectable by the

methods used here. This already represents

several thousands ofnewly synthesized viral

RNAmolecules (35).

The fact that a given RNA can direct the

synthesisofaknownproductinacell-free

pro-tein-synthesizing system, together with the

finding that the same RNA is associated with

polysomes,has beenregardedassufficient

evi-dence ofitsroleas amessengerinthecell. The

alphavirus 26SRNAfulfills both thesecriteria

and is regarded as the cellular messenger for

the structural proteins (4, 6, 7, 8, 14, 22, 27, 31, 32, 37). According to the same criteria, the 42S

RNAshould be regarded asthe messenger for

the nonstructural proteins; it has been found

associated with the polysomesininfected cells

(22, 27, 31), and it stimulates the synthesis of

nonstructural proteinsinvitro (5, 8, 9,. 28). It

wastherefore interesting to correlate the rate

ofsynthesis of the structural and nonstructural

proteins with the amount of their respective

messenger RNAs. The rate of synthesis of the

structuralproteins and the amount of 26S RNA

rose with almostidentical kineticsthroughout

the infection (Fig. 5C), suggesting that the

amount of structural proteins synthesized is

dependent on the amount of their messenger

RNA.This situation is similar to thatfound for

the synthesis ofa-and ,3-globinchains in the

reticulocyte lysate (34).

The rate of synthesis of the nonstructural

proteins correlated poorly with the amount of

their 42S RNA messenger.The maximum rate

ofsynthesis of these proteins was observed at

3.5 hpostinfection when only about half of the

42S RNAfoundat 5.5 hpostinfection had been

synthesized (Fig. 5B). Aroughestimateof the

efficiency of42S RNAat 3.5and5.5h

postinfec-tion canbeobtainediftheamountof

radioactiv-ityincorporated into the nonstructural proteins

during the 20-min pulse is divided by the

amount of radioactivity accumulated in 42S

RNA from 1 hpostinfection. There is about a

sixfolddrop in the "messengerefficiency" of this

RNAbetween 3.5 and 5.5 h postinfection.

One apparent possibility to explain the

re-duced messenger activity of the 42S RNA is the

consumption of this RNA in the formation of

viral nucleocapsids (11, 29, 32), which would

makethe 42S RNA inactive as messenger. The

maximum rate ofsynthesisofthe nonstructural

proteins taking place at 3.5 h postinfection

wouldthen be the result of two processes: the

synthesis of enough 42S RNA and the lack of

structural proteins, i.e., capsid protein to

en-capsidate this RNA. Later, when the rate of

synthesis of the structural proteins increased,

all or almost all the newly synthesized 42S

RNA would become encapsidated, and only

smallamounts ofnonstructuralproteins could

betranslated dueto"leakiness" of the

encapsi-dation process. This would explain why the

bulkof42SRNAsynthesizedbetween4and5 h

postinfection isfoundinthe 140Snucleocapsid

(27, 29, 32, 36). The process of association of the

capsid protein with the 42S RNAisseemingly

fastand, at 5 to6 h, postinfection takes place

within 5 to 8 min after the protein has been

synthesized (29).

The mechanism of nucleocapsid assembly is

poorly understoodatpresent. Ithas been

dem-onstrated recently that the SFVcapsid protein

bindsrapidly the60Sribosomalsubunit inboth

infected cells (36) andinvitro(10). The

signifi-cance of thecapsid protein-60S complexinthe

nucleocapsid assembly (11) andinthe control of

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149

the translation of the nonstructural proteins is

under investigation.

ACKNOWLEDGMENTS

Wethank RitvaRajalaandMizjaSalonen for excellent technical assistance. Actinomycin D wasagiftfromMerck, Sharp and Dohme.

This work was supportedby grants fromtheSigrid Juse-lius Foundation and the Finnish Academy. B.L. is a recipi-entofascholarshipfrom the Finnish Ministry of Education.

LITERATURE CITED

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Comple-mentationbetweentemperature-sensitivemutants of Sindbis virus. Virology 30:214-223.

4. Cancedda,R., and M. J. Schlesinger. 1974. Formation ofSindbis virus capsid protein in mammalian cell-free extracts programmed with viral messenger RNA. Proc.Natl. Acad. Sci.U.S.A. 71:1843-1847. 5. Cancedda, R., L. Villa-Komaroff, H. Lodish, and M.

Schlesinger. 1975. Initiation sites for translation of Sindbis virus 42S and 26S messenger RNAs. Cell 6:215-222.

6. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane proteins in alphaviruses. Nature (London) 254:454-455.

7. Clegg,J.C. S., and S. I. T. Kennedy. 1975. Initiation of the synthesis of the structural proteins ofSemliki forest virus. J. Mol. Biol. 97:401-411.

8. Glanville, N., J. Morser, P. Uomala, and L. Kkiri-ainen. 1976. Simultaneous translation of structural andnonstructural proteins from Semliki forest virus RNA in two eukaryotic systems in vitro. Eur. J. Biochem. 64:167-175.

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