La Crosse virus nucleocapsid protein controls its own synthesis in mosquito cells by encapsidating its mRNA.

0022-538X/89/125166-09$02.00/0

Copyright © 1989,American Society for Microbiology

La

Crosse

Virus

Nucleocapsid

Protein Controls

Its

Own

Synthesis

in

Mosquito Cells by

Encapsidating

Its

mRNA

DAVIDHACKER, RAMASWAMY RAJU,tAND DANIEL KOLAKOFSKY*

DepartmentofMicrobiology, University ofGeneva Schoolof Medicine, CMU, 9 Avenue de Champel, CH-1211 Geneva, Switzerland

Received 12 July1989/Accepted 24 August 1989

Within 24to48hof LaCrosse virus infection ofmosquito cells, >75% of the S mRNAwasfoundtoband in CsCl density gradients attheposition of genomeorantigenome nucleocapsids. Theencapsidation of the S mRNA correlates with therepression ofNproteinsynthesisinvivo,and theencapsidated SmRNA cannotbe translated in vitro. Unlike genome and antigenome assembly, S mRNA assembly isarelatively slow process, which is not coupled toits synthesis. Within the encapsidated S mRNApopulation, three forms could be distinguished, thosewith intact primers whichwere or were not also assembledwith Nprotein and those in which the primer and up to 3 template bases had been lost. We suggest that genome replication, but not transcription, is down regulated with time in mosquito cells for reasons that are unclear. The pool of unassembled Nprotein then increasedtothepoint atwhichitbeganto interact with its own mRNA, as this mRNAalsocontains what is considered to be the assembly site, i.e., the conserved sequencesatthe 5' ends of all genome andantigenome chains. This leadtotheassembly oftheentiremRNA, except forthenontemplate primer. Some of the primerswere then also assembledwithNprotein,whereasothersweredigestedtoproduce truncated mRNAs.

La Crosse virus (LAC) is a member of the California

encephalitisserogroupof theBunyaviridae(19). The genome

ofthese virusesconsists of three RNA segments of negative

polarity called L(large), M(medium), and S (small),which

encode the viralpolymerase (L), the surface glycoproteins

(Gland G2), and thenucleocapsidprotein (N), respectively

(16). The viral genomes (and antigenomes) are found as

helical nucleocapsids (NCs), assembled with the N protein,

in a structure which is sufficiently stable to survive CsCl

density gradient centrifugation. The minus-strand genomes

are templates for two kinds of RNA synthesis, that of mRNAs and antigenomes. mRNAs are initiated on capped

primers derived fromhost mRNAsbyacap-snatching

mech-anism (2, 17), whereas antigenomes (and genomes) initiate

withATP at theprecise 3' end of the template.

These viruses productively infect both mammalian and

insectcells, but the infection in these twocelltypesis quite

different (15). The infection of mammalian cells is highly

cytopathicandleadsinevitablytocelldeath,whereas thatof

mosquitocells is asymptomatic and becomes persistent. To

investigate the reasons forthe very different fates of these

infections,wehavecomparedBHKandC6/36cell infections

underotherwise identical conditions of cell growth (24). In

BHKcells,theinfection is relativelyrapid,and thelevels of

viral mRNAs and genomes reach a maximum around 6 h

postinfection (p.i.). At this point, the infection induces a

general mRNAinstability, to the extent that both host and

viral protein synthesis are severely reduced (22). New ge-nomesynthesis is also reduced at this time, and cytopathic effectbecomes visible by 14 h. Intheinfection of mosquito

cells,ontheotherhand, replication takes place more slowly

andtheintracellularlevels of viral RNAs and proteins reach

amaximumaround24h. However, the levels of viral RNAs

andproteinsinmosquito cellsatthis time often exceed those

*Correspondingauthor.

tPresent address: DepartmentofMicrobiology, Schoolof Med-icine, Washington University, St. Louis,MO 63110.

in mammalian cells.Thissuggeststhatcytopathic effectsare

not simply related to the accumulation ofviral

macromol-ecules (24).

All models for RNA virus replication are exponential

ones. Inthesemodels, primarytranscriptionandtranslation

allowafirst round ofgenomereplication,and theseamplified

genomesleadto anincreasedrateof viral mRNAandprotein

synthesis,which leadstofurthergenomereplication, andso

on.However,apointmustbereachedatwhichone or more

cellularcomponents become limiting, leadingto

cytopathic

effects.Yetthisclearlydoesnot occurduring LAC infection

of mosquito cells. Instead, LAC appears to limit its own

replicationingoing fromanacute(upto24h)to apersistent

phase(after24h) oftheinfection. Thesynthesis ofNprotein

andgenome replicationwerefoundto bemarkedly reduced

around24 h(24). Sinceacontinuoussupply ofunassembled

Nprotein is thoughttoberequired forgenomesynthesis,the

LACinfectionmaybecome self-limitingthrough the

unavail-abilityof Nprotein for assembly. Themechanismby which

N protein synthesis is repressed would then appear to be

central to our understanding of how the

mosquito

cell

infection becomes self-limiting.

The decline in N protein synthesis ataround 24 h is not

due to a reduction in the S mRNAs present, since their

steady-statelevels remainhighuntil72h. Infact,N

synthe-sisbeginstodeclineat atime when the maximum levelsofS

mRNAarejustbeing reached (24). Itthereforeappearsthat

translational control of S mRNA takes place in mosquito

cells. This reportsets out to examine this controlby

moni-toring the fate ofthe S mRNA during infection. We have

found that N protein controls the translation of its own message by assembling it into a NC. In addition, we have

investigatedthisassemblyasaway tobetterunderstand the

encapsidation of viral RNA.

MATERIALS ANDMETHODS

Analysis of cytoplasmic RNAs. LAC infections of C6/36

Aedes albopictus (8) and BHK-21 cells were carriedout at 5166

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33°C

(24). The

isolation

of NC RNA and unencapsidated

RNA

from

infected cells has also been describedpreviously

(20). For the isolation of intact NCs, the visible NC band

from a CsClgradient was removed through the side of the

centrifuge tube with a syringe. Recovered, NCs were

dia-lyzed for 3h inthepresenceof50 ,ug of tRNA per ml against

10 mM Tris hydrochloride (pH

7.5)-100

mM NaCl-1 mM

EDTA. TheNCs werepreservedin

10o

glycerol at -20°C.

ForNorthern(RNA) blot analysis,25

pRg

ofRNA that had

been pelleted through CsCl (CsCl-pelleted RNA) or the

cell-equivalent

amountof NCRNA washeated for2min at

90°C in a solution containing 80% formamide, 0.1% xylene

cyanol FF, and

0.1%

bromophenol blue and then cooled

quickly

onice.Electrophoresison2.8% polyacrylamide-8M

ureagels (1.5mmthick) inTBE(89mMTris-borate,89 mM

boric

acid)

was followed by electroblotting to Hybond-N

(Amersham)

in TAE (40 mM Tris-acetate [pH 7.8], 2 mM

EDTA).

Forslot blot

analysis,

1, 5,or25 ,ugofCsCl-pelletedRNA

orthe

cell-equivalent

amount of NCRNA wasmade 1 Min

ammonium acetate and then blotted to Hybond-N, using a

Schleicher and Schuell Minifold II.

Hybridizationswerecarriedoutin asolutionconsistingof

50%

formamide,

Sx SSC(lx SSC is0.15 MNaClplus0.015

Msodium

citrate),

and lx Denhardt solution with 5 x 106

cpmofprobe. Hybridizationswere conductedat50°Cwhen

the Sminus-strandriboprobewasused and at42°Cwhen the

cDNA

probe

was used.

Preparation of cDNA probe. C6/36 cells from 20

100-mm-diameter culture disheswere

lysed

in4mlofasolution

consisting

of10 mM Tris

hydrochloride

(pH 7.5), 100 mM

NaCl,

1 mM EDTA, and 0.5% Nonidet P-40. Nuclei and

cellular debriswereremoved

by centrifugation

at4,000 x g

for5 min. Total

cytoplasmic

RNAwasisolated

by

centrifu-gation through

a20to40% CsCl

gradient.

After

suspension

in TE (10 mM Tris hydrochloride [pH

7.5],

1 mM EDTA),

poly(A)+

RNA was isolated

by chromatography

on

oli-go(dT)-cellulose

as

previously

described(14).

For the

synthesis

ofacDNA

probe,

1,ugofpoly(A)+RNA

wasmixed with 6 ,ugof randomdeoxynucleotides

(6-mers),

denatured at

90°C

for 2min, and

precipitated

with ethanol.

The

following

reagents were used in the reverse

transcrip-tion reactranscrip-tion: 25 mM Tris

hydrochloride

(pH

8.3),

10 mM

MgCl2;

75 mM

KCI,

30 mM

P-mercaptoethanol,

500 ,uM of

dGTP, dATP,

and TTP; 10

uCi

[ot-32P]dCTP

(Amersham;

3,000

Ci/mmol;

10

mCi/ml);

and 10 U of

Moloney

murine

leukemia virusreverse

transcriptase.

Thereaction mixwas

incubated for30minat

37°C.

After the addition of cold dCTP

to aconcentration of250 ,uM, the reactionwas allowed to

proceed for another 30 min. RNA was thenhydrolyzed

by

heating

at 65°C for 1 h in the presence of50 mM NaOH.

After neutralization, the reaction mix was extracted with

phenol,

and

unincorporated

nucleotides were removed

by

gel

filtration.

Immunoselectionof RNA. For each

sample

tobe

analyzed,

40

RI

ofa50%

slurry

of

protein A-Sepharose

wasincubated

with 10

,ul

of either anti-NC serum or acontrol serumfor2 hat4°C. After three washes with coldNET(150mM

NaCl,

50mMTris

hydrochloride [pH 8.0],

0.1%Nonidet

P-40),

the beads were incubated with 100

RI

of

cytoplasmic

extract

(equivalent

to half of one 100-mm-diameter dish of C6/36

cells)

for1hat

4°C.

After three washes with

NET,

thebeads

were suspended in 0.3 M ammonium acetate (pH 5.4) and

0.5% sodium

dodecyl

sulfate. After

phenol

andchloroform

extractions,

the RNAwas

precipitated

with ethanol. North-ern

analysis

was carriedout as describedabove.

Invitro translation. Invitrotranslation was performed in

rabbit reticulocyte lysates (Promega) as recommended by

the supplier. The reaction products were analyzed by

so-diumdodecyl sulfate-polyacrylamide gel electrophoresis and

viewedby fluorography.

Primer extension. A gel-purified oligonucleotide comple-mentarytopositions44to57of the Santigenomewas5'end

labeled with [-y-32P]ATP (Amersham; 5,000 Ci/mmol; 10

mCi/ml) by using T4 polynucleotide kinase (Genofit). The

labeledprimerwasextractedoncewith phenol-chloroform, and 5to10pmol of labeled primerwasmixed with either20

,ugofCsCl-pelleted RNA or the cell-equivalent amount of

NC bandRNA,

precipitated

withethanol,suspendedin 6 ,lI

ofTE, heated for 2 minat 90°C, and brought to 0.2 M in

NaCl. Annealing was carried out at 29°C for 5 min, and

reverse transcriptase buffer was added to the following

reagents: 50 mMNaCl, 50 mMTrishydrochloride (pH 8.3),

8mMMgCl2,35 mM ,-mercaptoethanol, 500,uM

deoxynu-cleosidetriphosphates, and 100 U ofMoloney murine leu-kemia virusreversetranscriptaseperml inafinal volume of 30 pLI. The reaction was carried out at 43°C for 1 h and

stopped bythe addition of EDTA to10 mM. After ethanol

precipitation, the reaction products were suspended in a

solutioncontaining80%formamide,0.1%xylene cyanol FF,

and 0.1%

bromophenol blue,

heated at

90°C

for 2 min,

cooled on ice, and analyzed by electrophoresis on a 10%

polyacrylamide-8Mureagel (0.3mmthick).

RESULTS

Translational control of S mRNA by N protein.

Transla-tional control can be exerted at almost any level of the

translationprocess. For LAC S mRNA, it is

unlikely

tobe

ribosomal

loading

on the mRNA, since the 5' ends ofthe

viral mRNAs are derived from host mRNAs (2, 17). The

control mechanism also appears to be

specific

for viral

mRNA, sincecellular

protein synthesis

remains

unchanged

during

the

period

in whichN

synthesis

declines

(24).

One

possible

mechanism for this

specific

controlisthat N

protein

interacts with itsownmRNAtopreventitstranslation. The S genome segment is 983 nucleotides (nt) long. Its mRNAstarts on a

primer

of around15 ntand terminates at

position

886 and is therefore around 900nt

long.

In infected BHK

cells,

a minor

transcript

which starts at

position

+1

and terminates at 886

(hereafter

referred to as the 1-886

transcript)

hasalso been detected

(20). However,

unlikethe S

mRNA,

the 1-886

transcript

is found

only

intheform ofa assembled NC and must therefore contain the site for the

initiationof NC

assembly.

As the S mRNAcontainsall the

sequencesofthe 1-886

transcript,

it should alsocontainthis site.This situation is

unique

tothe

segmented

minus-strand RNA

viruses,

since their

nonsegmented

counterparts have thesiteofNC

assembly

on aleader sequencewhichis absent from the viral mRNAs(3).

TodeterminewhetherSmRNAisassembledinto NCsin

mosquito cells,

we

separated

NCs from

unencapsidated

RNAby sedimentation in CsCl density

gradients.

The dis-tribution of S mRNA between these two fractions was

determinedbyNorthern (RNA)

blotting (Fig. 1A).

Remark-ably, by

24h, 75%or moreof the intracellular S mRNAwas

found toband in CsCl at the

density

of NCs. The ratio of

unencapsidatedto

encapsidated

mRNAthenremained

con-stant to at least 72 h. In contrast, less than 10% of the S mRNAwas

encapsidated

at12h.Inaseparate

experiment

in

whichshorter time intervalswere

used,

the

majority

ofthe S

mRNA became

encapsidated

in a

relatively

short

time,

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A

hpi 12 24 72 B P B P B

*

*e

&w _ _

(D

CL

c

D

L

B

hpi

m BHK

2 3 C

probe S(-) T - +

12

~

24 _ , Mg

48 __ mD

72 _

BHK

12

24

48

72

ant,genome-:

-mRNA-+ l

IX 5X 25X

FIG. 1. AnalysisofencapsidatedRNA from C6/36 cells. (A)CsCl-pelleted (P)and band(B)RNAs fromLAC-infectedC6/36 cellswere

isolatedatthetimesindicated(hours p.i. [hpi]). Either 25 jig ofpelletRNAorthecell-equivalent amountof band RNAwasanalyzed by

electrophoresison adenaturing2.8%polyacrylamide gel.An S minus-strandriboprobewasused forhybridization. Thepositionsof theS antigenomeand mRNAareindicated. (B)Either 1, 5,or25,ugofCsCl-pelletedRNA(lanes 1, 2,and3)orthecell-equivalentamountofCsCl bandRNA(of thesamesamples usedabove)wereblottedontoaHybondNmembrane andhybridizedwithacDNAprobetouninfectedC6/36 poly(A)+RNA. BHKreferstoRNA fromLAC-infectedBHKcells whichwasisolatedat18hp.i.;mreferstoRNA from mock-infectedBHK

cells.(C) Immunoprecipitations ofNprotein-RNA complexesfromcytoplasmicextracts(18hp.i.)werecarriedoutasdescribed in Materials andMethods, usingantiserumtotheLACNprotein (+)or acontrolserum(-).Immunoselected RNAwasanalyzedon adenaturing2.8% polyacrylamide gel alongwith totalcytoplasmicRNA(T)fromanequivalent volume of theextractused forimmunoselection. RNAwas

blottedontoHybondN,andhybridizationwascarriedoutwith eitheranS minus-strand[S(-)] riboprobeorcDNAtoC6/36poly(A)+RNA. between 20 and 24 h (results not shown). The timing of S

mRNAencapsidation thus correlateswith theinhibition of N protein synthesis seenearlier(24).

To determine whether, in fact, encapsidation ofS mRNA preventsitstranslation, wecomparedthe levelof Nprotein

synthesis in a reticulocyte lysate programmed with the encapsidated RNA beforeorafterdeproteinization (Fig. 2). The deproteinized RNA was capable of synthesizing N

protein, whereasthe assembledRNAcouldnotbe translated at all. The N protein therefore appears to regulateits own

synthesisinmosquito cells, byencapsidating its mRNA and preventing its translation.

Specific encapsidationof viral mRNA. Hostprotein synthe-sis is notaffected byLAC infection ofC6/36cells(24).The assembly of mRNA into NCs would then presumably be specific for viral mRNA. To determine whether cellular mRNAs were encapsidated, 32P-cDNA was made from

uninfectedmosquito cell poly(A)+ RNAandusedas aprobe

against CsCl-pelleted and band RNA from infected cells. The cDNAprobe hybridized only to CsCl-pelleted RNA in slotblots (Fig. 1B), and identical results were obtained by

Northernblotting (results not shown).

From theabove experiment, however, it could be argued that Nprotein might have interacted with host mRNA but that the assembly was incomplete. Partially assembled

mRNAswouldnothavebandedatthe position ofNCs (and mightevenhave pelleted) and wouldnothave beendetected in the experiment shown in Fig. 1B. We therefore usedan

alternative approach which did not depend on complete

encapsidation. N protein-RNA complexes were

immunose-lected from infected cell lysates with anti-NC antibodies

(Materials and Methods). As a control, immunoselection

wasalso carriedoutby usinganantiserumtoaSendaivirus

protein. Northernblots of immunoselected and totalcellular RNAwereprobed with eitheranS minus-strand riboprobe orthemosquitocDNAprobe (Fig. 1C).Asexpected,viralS

antigenomes and mRNA were selected by the anti-NC serum, butnotby the control serum. Immunoselection was

not complete, however, as can be seen by comparing the

level ofantigenome in the total fraction with that of the immunoselected RNA. Incontrast toSmRNA,littlecellular RNAwasfound in the immunoselected fraction(Fig. 1G). A very small amount of RNA was immunoselected by both antisera, which probably represents interactions with the protein A-Sepharose. As our cDNA probe may only have been able todetect the more abundant mRNAs, wecannot rule out the encapsidation of some rare mosquito cell mRNAs. Within this limitation, however, the above results demonstrate that mostly viralmRNAs interact with N

pro-teinor areassembled intoNCs.

Asignificant poolof unassembled NproteininC6/36cells.

Since <10% of the S mRNAs are encapsidated at 12 h whereas the majority are encapsidated at later times, we

investigatedwhether thisdifferencewasassociated with the

intracellular level of unassembled N protein. If genome

synthesis andassembly are coupled, this level canbe esti-matedby measuringthefraction ofgenomesynthesiswhich continues after the inhibition of further protein synthesis. DuringBHKcellinfection,this levelappearstobeverylow,

inthat nogenome synthesiscanbedetectedby [3H]uridine

labeling after inhibition of protein synthesis (18). When similar experiments are carriedoutduring C6/36 cell

infec-p

- 4 mRNA

cDNA T - +

C1

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A B

N-

_

FIG. 2. In vitro translation ofencapsidated RNA. NCs were isolated frominfected C6/36 cellsat24handdialyzedto remove the CsCl. Rabbit reticulocyte lysatewasprogrammedwith either250 ng of the encapsidated RNA (B) or 250 ng of the same RNA after deproteinization(A).The[35S]methionine-labeledtranslation prod-uctswereseparated by15%polyacrylamide gel electrophoresis. The position ofNprotein,asdeterminedbyCoomassie blue stainingof aparallel nucleocapsidsample, is indicated.

Sample:

L L L L M M B

Drug: - a c P -P

M.,L

genomes_-S

genomes,,

-.

S

-MRNA-'"w

I

tion, the results are quite different. At 16 to 17 h, in cells

pretreated for 10 min with either anisomycin, cyclohexi-mide, orpactamycin, and in which [35S]methionine incorpo-rationwasreduced by >97% foreither 15or60min(results not shown), S genome synthesis continued at22to40% of the untreated controllevel (Fig. 3A). In these experiments, all of thepulse-labeled S mRNA pelleted through the CsCl

gradient, whereas all thegenomes werefound asassembled

NCs (resultsnotshown). Whenlabelingwascarriedout at20 to24h, 33to51% of S genomesynthesis continued (results notshown). However,when the experimentwascarriedout at5to7hp.i., genome synthesiswasnowstrongly reduced (3to6%of control [Fig. 3B]) but still clearly detectable. This experiment appears to measure the preexisting pool of unassembled N protein. When the [3H]uridine was addedat 1 h and 10min after drug addition rather than 10 min later and the cellswerelabeledat6to8 h(Fig. 3B),newgenome synthesis was undetectable. The ability of C6/36 cells to synthesize S genomes in the absence of on-going protein synthesis thus appears tobe duetotheir levelsof unassem-bled Nprotein, which increase with time. We note that these experiments donotestimateabsolute levels of N protein, but ratherthe levels required to support a given rate of genome synthesis, and that this rateis muchhigher in BHK than in

C6/36cells(24).

We also note that synthesis of the M and L genomes, which are respectively 4.6 and 7 times longer than S, is slightlymoresensitivetothetranslational block than that of S (Fig. 3). This suggests that the larger genome segments may replicate more slowly than S when the level of unas-sembled N has been reduced. It also offers a possible explanation for the curious finding that only the S segment

pulse

time:

5-7 6 E

0 0 oC

C-CHX:

o o o o c

IIcv

7

M,

L genomes *

I

I

|

~~~S-genomes

-o

4--S-mRNA 4m

FIG. 3. Asignificantpoolof unassembled N protein in C6/36 cells. (A) Infected C6/36 cultures were either not drug treated (-) or were pretreated for10 minwithaconcentration of100,ug/mlof cycloheximide (c) or anisomycin (a), or with 5 ,g ofpactamycin(p) per ml and thenlabeledwith 300,uC of[3H]uridine(26Ci/mmol)for 1 h. Thevisible NC band was isolated, and its RNA wasseparatedon adenaturing 4%polyacrylamide gel. Samples markedM arefrommock-infectedcultures;thosemarked LareLACinfected.Thesample markedBis total cytoplasmicRNAfrom infected BHK cellslabeled4to 5hp.i., run as markers. (B) Infected C6/36 cultures wereeithernottreated or treated with 100 or 200 ,ugof cycloheximide(CHX) per ml at 4 h and 50min. [3H]uridinewas then added at either5to 7 or 6 to 8 h, as indicated.

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hpi

Mock

B P

10-12 B P

22 -24 22 -26

B P B P

(J-ant

igenome

-mRNA

FIG. 4. Pulse-labeling of LAC RNAinC6/36 cells. LAC-infectedC6/36 cellswerelabeledwith 300,uCi/mlof[3H]uridine (26 Ci/mmol)at the times(hoursp.i. [hpi]) indicated.CsCl-pelleted(P) and band (B) RNAswere prepared and analyzed on adenaturing 2.8%polyacrylamide gel. After fluorography,the gel wasvisualizedby autoradiography. Thepositions of S antigenomes (andgenomes)andSmRNA areindicated. The bandsat the top presumably representthe M and L genomes andantigenomes.

canbe detected inlong-term persistently infectedmosquito

cellsortheparticles they shed (7, 24).

Synthesis and assembly of S mRNA are not coupled.

As-semblyof viral antigenomes and genomes into NCs occurs

either concurrently with their synthesis or shortly

after-wards, as these RNAs are found only in NCs. To determine ifthe same is true for the assembly of S mRNA, infected

cellswerepulse-labeledwith [3H]uridineateither10 or 22 h

p.i. (Fig. 4). If mRNA which was synthesized during the

labeling period wereconcurrently assembled, then like

ge-nomeandantigenome RNA, it shouldimmediatelyappearin

the CsClbandfraction.Ifassemblyoccurred after synthesis,

the mRNA should appear, at least partially, in the pellet

fraction. All of the pulse-labeled mRNA was found in the

pellet, whereas all of the genomes and antigenomes were

found in the CsCl band, regardless ofthe time oflabeling

(Fig. 4).Northern blotanalysis (resultsnotshown) indicated

thatencapsidationofthe S mRNAdidoccurasusualduring

this infection. Encapsidation of the S mRNA is therefore

clearly different from that ofgenomes and antigenomes in

that it is completed wellafter synthesis ofthe mRNA.

Mappingthe5' ends of encapsidatedRNAs.The 5' ends of

encapsidated S mRNA from BHK cells have previously

been mapped by primer extension. The encapsidated S mRNAwasfound to contain the same range of nontemplate primers (10 to 18 nt in length) as those on the unencapsidated S mRNA (20). In aneffort to understand why such a large fraction of the S mRNA was encapsidated in C6/36 but not in BHKcells, the 5' ends of encapsidated and unencapsidated S mRNA from mosquito cells were similarly examined,

along with RNA from BHK cells (Fig. 5). The results from

the BHK cellRNAweresimilar to those found previously.

TheCsCl-pelletedfraction (lane 8) contained predominantly

RNAswhose 5' ends were mapped to position -10 to -18,

witha minoramountof RNA whose ends were at position

-1to +4. TheCsCl bandRNA(lane 5),onthe otherhand,

contained onlyaminoramountofmRNAwithnontemplate

primers as well as antigenomes. However, two bands

ap-peared at the position corresponding to the 5' end of the

antigenome. To determine which extension product

termi-natedatposition +1, asequenceladderofthis endof theS

segmentwasproducedfromafull-lengthclone(4). Atits 5'

end,theantigenomesequenceis5'AGTAGTGTACTCCAC

3',and itscomplementis marked inFig. 5.By comparison,

the upper band of the doublet was found to represent

position +1 (marked bya dot). Thereasonfor the band at

position +2 is unclear. The same reverse transcriptase

yieldeda single band atposition +1 onextensionto the 5'

end of genome from the same RNA sample (results not shown), eventhough the last 27 nt of genomes and antige-nomes are virtuallyidentical.

When the C6/36 cell RNA was examined, the

pelleted

fraction(Fig. 5, lane7)wasfoundtocontainRNAwhose 5'

ends wereidenticaltothose in BHKcells (-10to -18nt),

except that there were even less RNAs whose 5' ends

mappedtoposition-1to+4.However,primer extensionon

theencapsidatedS RNAsproduced unexpectedresults(lane

4). Products which extended10 to18ntbeyondposition +1, as in the pelleted fraction, were very abundant here, but therewerealsonumerousbandsnearposition +1insteadof thedoublet. Fivemajorbands whichcorrespondedto

posi-tions -1 to +4 of the antigenome were present. To deter-mine if these bandsrepresentedfurtherheterogeneityatthe 5'endof theantigenomeoriftheyweretruncatedspeciesof mRNA, S mRNA andantigenomeRNAfrom theCsClband were isolated froma 2.8% polyacrylamide gel(cf. Fig. 1A)

and analyzed separately. Extension on the

antigenome-length RNA (lane 1)producedthesame twobands (+1 and

+2) as observed for antigenome RNA from BHK cells.

Extension onthe isolated mRNA (lane 2) revealedthat the

heterogeneityaroundposition+1resulted from the presence

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LAC S mRNA ENCAPSIDATION 5171

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1 2 3 4 5 6 78

FIG. 5. Mapping the 5' ends of encapsidated S RNAs. CsCl-pelleted and band RNAs from LAC infections of BHK(8 h p.i.) (lanes 5 and 8) and C6/36 cells (24 h p.i.) (lanes 4 and 7) were

examinedby primer extension (see Materials and Methods). The reaction products were analyzed on a 10o sequencing gel. CsCl band RNA from C6/36 cellswasalsoseparatedon adenaturing2.8% polyacrylamide gel. After ethidium bromidestaining,Santigenome (and genome) RNA and S mRNA were isolated separately and analyzed byprimerextension(lanes1 and2).On the left is shown the sequence produced by the dideoxynucleotide triphosphate

method fromafull-lengthSclone, usingthesameoligonucleotide.

Thesequencecomplementarytothe5'end of the Santigenomeis indicated, with the first virally encoded nucleotide (position +1) marked with a solid circle. The poly(C) tail used in the original cloning is visiblejust above. The positions of the 5' ends of the nontemplateprimersfrom S mRNA(-10to-18)and thepositionof the5'end of the Santigenome(+ 1)areindicated. Position+1of the Santigenome is also indicatedbyasolidcircle in lanes 1 and 5.

of truncated mRNAspecieswithin theencapsidatedmRNA population.

Truncated mRNAs. We nextinvestigated when the trun-cated mRNAs appeared during the infection, by primer extensiononRNAsfrom various timepoints (Fig. 6A). For theCsCl bandRNA,the doublet band from theantigenome (position +1 is marked by a solid circle) was the major species present at the earliest time when viral RNA was

visible(11 h).Averylow level of intact mRNAwasalsojust visible,butwe wereunabletodetect bandsatpositions -1,

+3, and +4,

i.e.,

evidence of the truncated mRNAs. With

time,

the levels of

encapsidated mRNA,

both intact and

truncated,

increased. However, the level of the truncated

mRNAcontinued toincrease upto 72

h,

whilethat ofthe

intactmRNA remained constant after 48 h. For the

CsCl-pelleted RNA,

thelevel of S mRNA reachedamaximumat 24 handthenfell

sharply by

72h.As

before,

the truncated

mRNAswereveryminor constituents of this fraction.

Thus,

not

only

arethe truncated mRNAs found almost

exclusively

in

NCs,

but thetimecourseoftheir appearance suggests that

they

are derived from the

encapsidated

mRNAs whose

primers

areintact.

When thesesameRNA

samples

were

analyzed by

North-ern

blotting,

it could also be seen that the RNA

species

presentintheCsCl bandhad

changed

with time

(Fig. 6B).

At 11

h, only

antigenomes (marked by

a solid

triangle)

were

visible,

and

by

24

h,

averysmallamountofmRNA

(marked

by

asolid

diamond)

also

appeared

in the CsCl band.

How-ever, at 48 and 72

h,

besides a considerable amount of

mRNA,

athird

species (marked by

asolid

circle) just

below

that of the intact mRNA

appeared.

This

species,

whose

migration

is consistent with an RNA that is 15 to 20 nt shorter than intact S

mRNA,

is

presumably

the truncated

mRNA observed

by

primer

extension

(Fig. 6A).

A similar

RNA

species

has also been seen

relatively

late in BHK

infections,

but its5' endwas

mapped

to

position

+1 without

heterogeneity,

and itwasreferredtoasthe 1-886 RNA

(20).

Wealsonotethat the

encapsidation

ofthe

majority

ofthe S

mRNAin this infection occurred

slightly

later

(between

24

and 48

h)

thanthat shown in

Fig. 1, possibly

becausealower

multiplicity

ofinfectionwasused.

The

primer

onviralmRNAcanalso become

encapsidated.

The

finding

that

only

viral RNAs are assembled into NCs suggests that a

specific

RNA sequence or structure is

re-quired.

If

only

alimited sequence were

involved,

themost

likely assembly

site would be the

highly

conserved 11 nt

(AGTAGTGTACT)

atthe 5' ends ofall three genomes and

antigenomes (20).

Asthere isalso

clearly

an

assembly

siteon the S

mRNA,

the

question

then arises whether the 5'

nontemplate

primer

can also become

encapsidated.

The interest here concerns whether

encapsidation

canbe

bidirec-tional,

or whether it occurs

only

in the 5'-to-3'

direction,

having

initiatedatthe conserved 5'ends ofthechains.

If the

primer region

of themRNAwerealso

encapsidated,

then it would bemoreresistanttonuclease

digestion

thanthe

samesequenceson

unencapsidated

mRNA.NCswere

there-fore isolated from

mosquito

cells

by

CsCl

banding, dialyzed,

and treated with

increasing

concentrations of either

micro-coccal nuclease

(MNase)

orRNase A inthepresence of400

,ug of

pelleted

RNA from uninfected cells per ml. Similar

results were obtained with both

nucleases,

and

only

those with MNaseareshown in

Fig.

7. On the

right

areshown the

extension

products

from untreated BHK cell

CsCl-pelleted

and band RNAs for reference. MNase treatment of the

mosquito

cell

pelleted

RNA showed that >90% of this

mRNAwas

digested

with 5 ,ug of MNase per ml. At

higher

concentrations,

all the mRNAwas

digested.

Incontrast, 25

to35% ofthe

nontemplate

primers

onthe

capsidated

mRNA

wereinsensitiveto asmuchas25,ug ofMNaseper ml. There also

appeared

to be a

population

of

encapsidated

mRNA whichwas sensitivetoMNaseandwas cleaved

by

concen-trations of 5

,ug/ml

or more. Also present in the CsClband

fractionwerethetruncated mRNAs

(positions

-1to

+4)

and

antigenomes (+1

and

2).

None of these bandsdecreased in

intensity

throughout

the range ofMNase orRNase A

con-centrationsbutinstead increased

slightly.

This increasemay

VOL.63, 1989

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B

-NC

Band

-

r

-

Pel-let

m 8 11 24 48 72 m tt 24 48 72

hpi

1

1

24

4

8

P B

P

B

P B

as o

ve

0

[image:7.612.74.548.79.368.2]

to IN

FIG. 6. Timecourseofappearanceofthe truncated SmRNA.(A) CsCl-pelletedandbandRNAswereisolated from LAC-infectedC6/36 cells atthe times (inhours p.i. [hpi]) indicated (m refersto mock-infected cells) and analyzed by primer extension (see Materials and Methods).Thepositions ofthenontemplate primerontheSmRNA (-10to-18)andthe 5' end oftheS antigenome(+ 1)areindicated. (B)

TheCsCl-pelleted(P)and band(B) RNA fromthesameexperimentwereseparatedon adenaturing2.8%polyacrylamide gel usingeither 25 p.gofpelletRNAorthecell-equivalentamountofbandRNA. The RNAwasblottedontoHybondNandhybridizedwithanS minus-strand

riboprobe. The positions of the S antigenome (A), S mRNA(*), andtruncatedSmRNA(0)areindicated.Anunderexposure of thisblotis shownsothatthedifferenceinmobilitybetween the S mRNA andthe truncatedmRNAisvisible.

haveresulted fromsomeof the intact mRNAswhich specif-ically lost their primers.

Insummary, wefoundthat inasizeableproportion ofthe mRNAs which band atthedensity ofgenomeNCs inCsCl

(30to50%asjudged byMNaseand RNase Aresistance),the

nontemplate primerswereasresistanttonuclease attackas

the first55ntof the templatesequence. On these SmRNAs,

NCassembly musthaveoccurred bidirectionally. DISCUSSION

During LAC infection ofmosquito cells, an initial acute phase of virus replication evolves into a noncytopathic

persistent infection, asthe virusdown regulates its

replica-tion (24). One aspect of this control is the inhibition ofN protein synthesis, by the binding of N protein to its own

mRNA. The N protein-mRNA interaction was found to be highlyspecific forviralmRNAs, and assembly occurredon

performed mRNAs, rather than concurrently with their synthesis. Theencapsidated mRNA was comprised oftwo populations, those whose primers were intact and those whose 5'endswerelocatedatpositions -1 to +4. Examin-ing mRNAs with intactprimers, we were surprised to find

some in which these sequences were also encapsidated. Theseresultsarediscussedbelowintermsof the persistent infectionand their relevancetothe NC assemblyprocess.

Translational controlofNsynthesis. Several examples of translational controlby mRNA-binding proteinshave been

described. Ribosome scanningof ferritin mRNAs is attenu-atedby thespecific bindingofacytosolic proteintoa35-nt

sequence in the 5' untranslated region (13, 25). Stored mRNA in Xenopus oocytes are silent due to interactions with oocyte-specific mRNA binding proteins (9, 23). In procaryotes, the T4 gene 32 protein (12, 26)and severalof the ribosomal proteins of Escherichia coli (5) are part of

self-regulatory systemsinwhich theproteinbinds withhigh affinityto its primarytarget(single-strandedDNAforgene

32proteinand rRNA for ribosomalproteins)and with lower affinitytoitsownmRNA. For theribosomalprotein S4,the site in the 5' untranslatedregion ofthe mRNAmay have a

similarstructure tothebinding siteon therRNA(6), while the siteongene32 mRNA isanunstructuredregionnearthe startcodon (11).

The control of LAC N protein synthesis is somewhat similar to the latter twoexamples. N protein recognizesa

specific bindingsiteonits own mRNA,andoncethe initial interactions have taken place, further N binding would presumably become cooperative due to N-N interactions, andassemblywouldensue. Ribosomes, asexpected, could

not translate this complex in vitro. Because the N binding site ishighly specific,thiscontrol is limited eitherentirelyor

mostly to viral mRNAs. One otherexample ofa viral NC protein interacting with viral mRNA has been reported recently (27). The leader RNA of mouse hepatitis virus, whichacts as aprimerfor mRNA synthesis, is specifically

A

hpi

-18

-10

72

P

B

A.

mock E

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LAC S mRNA ENCAPSIDATION 5173

Pellet i-NC Band-- BHK

pg/ml

0 1 5 1025 0 1 5 10 25 B P

) -18

Noit.

= _ w _ S~~~~~~~~~~,_s~- -+1

__

.*r

._ .. .. .. _ _

FIG. 7. Micrococcal nuclease sensitivity of encapsidated S

RNA. NCs(NC band)andCsCl-pelletedRNA wereisolated from

LAC-infected C6/36cellsat24 hp.i. Before nucleasetreatment,0.4

mg of mock-infected C6/36pellet RNA per ml was added to the

NCs.When infectedpelleted RNAwasanalyzed, itwasalsopresent at 0.4 mg/ml. The RNA samples were treated with increasing

concentrationsof micrococcalnucleaseasindicated in thepresence

of 2 mMCaCl2.After the reactionwasstoppedwith 10 mMethylene

glycol-bis(P-aminoethyl

ether)-N,N,N',N',-tetraacetic acid(EGTA),

the RNAswereextracted and analyzed by primer extension. On the rightareshown inprimer extension products from untreated CsCl-pelleted (P)andband(B)RNAs fromLAC-infectedBHK cells. The

positionsof the5'ends of S mRNA(-10to -18)and the 5' end of theSantigenome (+ 1) areindicated.

bound by the NC protein. It has not been determined, however,ifthis interaction affects translation.

mRNA encapsidation. The encapsidation of viral mRNA hasprovidedanunexpectedwayinwhich NCassemblycan

be examined. Previously, it was inferred that N protein

interactedspecificallywith viralRNAs,asonlygenomesand antigenomesarefoundinNCs. The basis for thisspecificity, however, was unclear. It couldnot be ruledout, for

exam-ple,that N simply recognized RNAswitha5' triphosphate,

there being no other triphosphorylated RNAs in the cyto-plasm with which to compete. We have shown here that underconditions in which >75% of the viral S mRNA was

encapsidated, little or no cellular mRNA was found to be

even partially assembled. In this case at least, N protein must recognizeaspecificviral sequenceorstructure.

ItseemslikelythattheNprotein assemblysiteonmRNA is the same as that ongenomes and antigenomes, which is thoughttobe the conservedsequencesatthe 5' ends of these chains. Ifso,thissequencedoesnothavetobe locatedatthe precise 5' end of the RNA to be recongized. However, recognition of the assembly site is much less efficient for mRNAthanforgenomesandantigenomes,since encapsida-tion ofmRNA occurs at least 4 h after its synthesis. This suggeststhat N proteinbinds with a much loweraffinityto mRNAthan toantigenomes. The difference is presumably due to thenontemplate primer on the mRNA. This primer separatesthe conserved 5' endsequencesfrom the

triphos-phate group, and this 5' end group is now also blocked with am7G.

Among the encapsidated S mRNA, defined by their gel

migration (900 nt as opposed to 983 nt), we find two

populations: those whose primersareintact and thosewhose primers and upto3template bases appear to have been lost. The timecourseof the appearance of the truncatedmRNAs and their relative absence among the unencapsidated mRNAssuggeststhattheyarisefromencapsidatedmRNAs

which are intact. Nuclease treatment of the encapsidated

RNA eliminatesonlysomeof the intact mRNAs andslightly increases the population whose 5' ends mapatposition -1to

+4. The simplest explanation for the truncated mRNAs is

thatthemRNA is firstassembledcompletely except forits 5'

primer. Insomeof thesemRNAs,theprimerwould then be

encapsidated by assembly in the 3'-to-5' direction, and so

become resistanttonuclease attack.Assemblyin this direc-tionwould be less efficient than in the 5'-to-3' direction. In

the others, nuclease would attack the unassembled primer

anddigestthe 5' endtopositions -1to +4. This scheme is

consistent with the assembly site being the conserved 5' endsof the genome andantigenomechains.

Establishingapersistent infectioninmosquito cells. mRNA

encapsidationis highly cell-type dependent, since it occurs

to very different extents in BHK and C6/36 cells, and the reasons for this difference appear important for the

self-limiting infection of mosquito cells. During replication of

nonsegmented minus-strand RNAviruses suchasvesicular

stomatitis virus and Sendai virus, the level of genome

replicationiscontrolledbythatof unassembled Nprotein,as

genome replication and assembly are coupled (for recent

reviews, see references 1 and 10). This also appears tobe

truefor LAC in BHK cells(18, 21)andatearlytimes(5to8

hp.i.)in mosquitocells (Fig. 3B). By 16 hp.i. inmosquito

cells, however, 22 to 40% of genome replication still

oc-curred in the absence of new protein synthesis, yet the genomes which were made here were all encapsidated.

Theseresultsindicate thatapool ofunassembledNprotein

exists in C6/36 cells and that this

pool

increases with time.

When this pool increases, several dramatic changes take

placewithin theinfectedcells. These include therepression

of genome replication and N protein synthesis and the

encapsidation

ofmostof the S mRNA.

It is

paradoxical

that the rate of genome replication is maximalearlyinthe infection when thepoolof unassembled N

protein

is low andbeginsto decline at atime when this

pool

hasincreased. One

possible explanation

for this is that the S mRNAbecomes the

preferred

targetfor NC

assembly

overgenomes and

antigenomes

with time.

However,

newly

synthesized

mRNAremains

unencapsidated

upto4h after its

synthesis,

even at times when the level of mRNA

encapsidation is high. If its affinity for N protein had

changed,

we would have

expected

that the

newly

synthe-sized mRNA would have been more

quickly

encapsidated. We also know that S mRNA

synthesis

remains

relatively

constantupto5

days p.i.,

and

during

this

period

alowlevel

ofN protein

synthesis

is observed(24).

Given these

findings,

it is difficult to accept a model in which the encapsidation of S mRNA and a

subsequent

depletion of unassembled N

protein

are

responsible

for the

shutoff of genome

replication. Instead,

it would appear that genome

replication

is down

regulated

as a result of the

absenceofafactor other than unassembled N

protein.

In this

model, it is the lack ofnewgenome

synthesis

which allows

the

pool

of unassembled N

protein

togrow, sothat it now

interacts with its lower

affinity

target,theviral mRNA. This VOL. 63,1989

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[image:8.612.55.298.75.308.2]

leadsto adecline in N synthesis, whichcanbe viewedas a subsequent level of control in the persistent infection. If this secondary targetfor encapsidation didnotexist, onewould expect that the unassembled N would eventually interact with host mRNAs to a greater extent or become insoluble, leadingto cytopathiceffects.

The arrestof genome replication in mosquito cellsoccurs in the presence of both functional N and Lproteins. Enough N protein is available to encapsidate a large fraction of mRNA, and L protein is active, as demonstrated by the continued synthesis of viral mRNA. From these observa-tions, another factor may be required for genome replica-tion, and it is the absence of this factorwhich limitsgenome replication in mosquito cells. Such a factor would presum-ably be required for theinitiation of genome replication (i.e., initiation with ATP, rather than a primer) and could interact with the L protein to modify its activity from a transcriptase to areplicase.

ACKNOWLEDGMENTS

We thankCatherineStouderfor experttechnical assistance and RichardCompans for helpful discussions.

Thiswork was supported in part by the Swiss NationalScience Fund. D.H. was supported by a fellowship from the Fogarty International Center.

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