The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation.

JOURNALOFVIROLOGY, July1993, p.4358-4364 0022-538X/93/074358-07$02.00/0

Copyright C)1993,AmericanSocietyforMicrobiology

The Hypervariable C-Terminal

Tail of the Sendai

Paramyxovirus

Nucleocapsid

Protein Is

Required

for

Template

Function

but Not for

RNA

Encapsidation

JOSEPHCURRAN,' HORSTHOMANN,2CHRISTIANBUCHHOLZ,2SYLVIE

ROCHAT,1

WOLFGANG NEUBERT,2 ANDDANIELKOLAKOFSKY1*

Department of Genetics andMicrobiology, University of Geneva SchoolofMedicine, CMU, 9 Ave deChampel, CH1211Geneva, Switzerland,1and Max Planck InstituteofBiochemistry, Department

of Virology, Am Klopferspitz 18a, D-8033 Martinsried, Gennany2 Received 18February 1993/Accepted 20 March 1993

Theparamyxovirusnucleocapsid proteins(NPs)arerelatively well conserved,exceptfor theC-terminal20%o (orca.100aminoacids), referredtoasthetail. We haveexamined whether thishypervariabletail isrequired

forgenome synthesis, bothinvitro,where synthesisispredominantlyfrom theinput templates, and invivo,

wheremultiplerounds ofamplification occur.Intheseviruses,genomesynthesis andassemblyof thenascent chainarecoupled.We find thatthe tail isrequiredinvivo butnotinvitro. Closer examination of theinvivo systemshowedthat the tailless NP couldencapsidatethegenomechainbut thatamplificationdid notoccur.We

interpret these results as indicating that the tail isnot required for RNAassembly but is required for the

templatetofunction in RNAsynthesis.Relativelysmall deletions within the conserved N-terminal 80%oofthe

protein,onthe other hand, rendered the proteinnonfunctional ineithersystem.Thepossible functionsof the tail in RNAsynthesisarediscussed.

The nonsegmented genomes of paramyxoviruses (ca. 15

kb)arefoundpredominantlyashelicalnucleocapsids (NCs), inwhichthegenomeRNAistightlyassociatedwithca.2,600

copies of the viral nucleocapsid protein (NP [8, 18]). Two

other viralproteins, L (large) and P (phosphoprotein), are more loosely associated with NCs, and togetherthis

com-plexcansynthesize mRNAs from theminus-strandgenome. P and Ltogether are thought to constitute the viral poly-merase,whereas NPis consideredtobepartof thetemplate,

because thepolymerase willnot copynaked genome RNA (11, 12). Duringgenome replication,NP also assembles the nascent antigenome chain, and thisconcurrent assemblyis

presumably required to maintain the processivity of the

polymerasein traversingthetemplate (28). TheNP

respon-sible for this assemblyappears to actas anNP-P complex, possiblyin association with the P-Lpolymerase (15).

ThesequencesofnumerousparamyxovirusNPshavenow been predicted (reference 29 and references therein), and comparisonsof themcoupled with proteasestudiessuggest that NP can be divided into two domains. The N-terminal

80%of theprotein is relativelywellconservedamongrelated

viruses, whereas the C-terminal 20% is poorly conserved. This hypervariable C terminus appears to be a tail at the

surface of aglobular N-terminal body, because the Sendai virus (SEN)NP(62 kDa), forexample, ishypersensitive to trypsindigestion,leavinga48-kDaN-terminalcore(13, 24). The tailalsocontainsmostof theprotein'sphosphorylation

sites(16)andantigenic sites (2, 10). The tail's hypervariabil-ity suggests that it may notbe functionally important, and largepartsof thisregionwererecently foundtobe

dispens-ableforbindingto Pwithaninvitro blottingassay(14). Systems to study SEN genome replication, both in vivo and in vitro, have recently been described (3, 15). The in vivo system uses copy-back defective interfering (DI)

viri-*Corresponding author.

ons (freed of their helper virus by UV inactivation) to naturallyinfectcells,andthehelperfunctionsforreplication

areprovided bytransfection ofplasmids expressing NP, P, and L. The in vitro system uses core DI genome NCs (washed free of their P and L) as templates and crude extractsof transfected cells as asource of the viral helper proteins. We have studied whether the hypervariable C terminus of NP is required for its function in genome replication byusing both of thesesystems.

MATERIALS AND METHODS

Construction of DNA subclones. The construction of

pGEM-NPwth, pGEM-P/C, pGEM-L, and some of the NP deletion mutants has been previously described (3-5, 14).

Most of the NP deletion mutants detailed in Fig. 5 were cloned by fusion polymerase chain reaction. The PIV1 pGEM-NP clone (clone 4-31) was a kind gift from Yumi Matsuoka(21). The mutantpGEM-NPBalwasgeneratedby

fusing the uniqueBalIrestriction site (Fig. 1) to ablunted BamHI site in the downstreampolylinker.TheC-terminally

truncated protein expressed from this clone would contain two additional (non-NP) codons followed by an amber codon. pGEM-NPNco was constructed by insertion of a

three-way-stop oligomer, 5'CTAACTAGTI1G, into the blunted NcoI site. Thisduodecamer isperfectly palindromic

andcan be self-annealed to providedouble-stranded DNA for insertion intoanyblunted site. It contains stop codons in allthreereading frames,inboth orientations (oneis

under-lined), and itsSpeI restriction site (in boldface) allowseasy

screeningofclones.

Invitro and invivo RNAsynthesis.DIH4Uv amplification

in vivo was performed as described previously (3). RNA

synthesisinvitrowasperformed essentiallyasdescribedby

Curranetal. (6),with thefollowingmodifications. NP-RNA

(RNP) templates were prepared from egg-grown defective virus by first pelleting the virus through a cushion of25%

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co IT

92% (CR1) 47%

--- mL

mO z<0

co z

'9 L

C-0

11-28% 77% 16% 65%

CR2AMmCR3

II

%activity

98

±10

29 ± 4

<2

+ - + +

P1l Eq1 EEE t nDE DitDi1 nK iaR R1aDx R 491

SEN 1 R 1 EB E tn D EDv sDsi R Ri a mR1aE R R 491

bPI3 D tqt1 svt1 iBsiKtEqRn i R D R 1 nR R 481

hPI3 DD R t q at soDniKt Eq q n i R D R1nxR 481

_- -_+

FIG. 1. Sequence comparison of the SEN proteins and PIV1

NPs.Thetop boxshowsthehomologiesoverthe entire length of the

proteins.Thenumbersabove refertoresidueposition; the percent-agesbelowrefertoamino acid identities. TheN-terminal80% ofthe

protein ishighly conserved (CR1), whereas the C-terminal20% is

more divergent. Below, an enlargement of this divergent domain

reveals two blocks of higher homology, CR2 and CR3. Three restrictionsitesusedinthesestudiesto generateC-terminal

trunca-tions are also shown above. These mutants are diagrammed just

below, and their percent activity relative to NP' in genome

amplificationinvivo(Fig. 2) isindicated. Atthe bottom is shown a

four-wayalignmentoftheprimarysequencesof the CR2 domainsof SEN, PIV1 (PI1), hPIV3 (hPI3), and bPIV3 (bPI3). Positions in

which a positive or negative residue is conserved in all four sequencesareindicatedinboldface andwithanasteriskbetweenthe

two pairs. The plus and minus signs refer to conserved charged residueswithineachpair.

(vol/vol) glycerol in TNE (10 mM Tris [pH 7.4], 50 mM NaCl,1mMEDTA),resuspendingthe viralpellet in 150 mM NaCl-50 mM Tris (pH 7.4)-10 mM EDTA-0.6% (vol/vol)

NonidetP-40(lysis buffer), and thenbanding theNCs twice on 20 to40% (wt/wt) CsCl gradients (38,000 rpm for 2 h at 12°C in an SW41 rotor) to remove the endogenous

poly-merase. This material was then diluted threefold in water,

andtheNCswerepelleted by spinningat50,000rpmfor 90

minat12°CinanSW60rotor.Templateswereresuspended in20%glycerol-water (50 ,u/10ml of startingallantoicfluid) andwerestoredas10-,ul aliquotsat-70°C.Normally,2,ulof templatewas usedperreaction.

Cytoplasmic extracts were prepared essentially as

de-scribed previously (6), exceptthat CV1 cellswerereplaced

with A549 cells (a human epithelial linewhich gavehigher

levelsofgenomereplication invitro) and theactinomycinD

levelswere increased to afinal concentration of 20 ,ug/ml. Thiselevated concentrationwas requiredto inhibit all

T7-derived DNA-dependent RNA synthesis in the extracts (24a). Cells were infected with the vaccinia recombinant vTF7-3(9)atamultiplicity of infectionof 2 PFUpercell 30

min

before transfection and were harvested at 24 h postin-fection by solubilizing the monolayer in lysis buffer. NC and RNA products were separated on a 20 to 40% CsCl gradient. The NCs were phenol extracted and analyzed directly on a 1.5% agarose-formaldehyde gel.

Antibodies and probes. The monoclonal antibodies NP877 and P 1.180 were a kind giftfrom Claes Oervell, Stockholm, Sweden. The monoclonal antibody NP W16 (which

recog-nizes both Sendai

NP'W

and

NPBal

[10])

was akind giftfrom Alan Portner.The L monoclonal antibody was raised against aC-terminal peptide and has been reported previously (7).

RESULTS

The precise sequence of the C-terminus of the SEN NP has been the subject of some confusion. The sequences of the Z (27) and Enders (23) strains were reported first, and these werebasically identical except for asingle-base inser-tion or deleinser-tion within codon 493. The C termini were consequentlydifferentin sequence from this point, as well as different in length (strain Z was 524 amino acids [aaJ and Enders was 517 aa). Shioda et al. (27

[corrigendum])

cor-rected their sequence atthis site to agree with that of Morgan etal. (23), yielding one sequence of NP, which we refer to as PQQ-517 (PQQ are the last 3 aa of this sequence of 517 residues). In 1990, however, Middleton et al. (22) reported that the NP gene of their Z strain

corresponded

to that initially reported by Shioda et al., and essentially the same protein sequence was then reported by Neubert et al. (25)for

three separate variants of the Fushimi strain. In this last

case, moreover, the predicted C termini (all GGI-524) were

confirmed by the sequencing of tryptic peptides. It then seems likely that GGI-524 represents the

correct

C terminus of SEN NP, and we provide some evidence below thatthis is so. In retrospect, the original 1983 sequence contained the GGI-524 C terminus because it also contained a nearby compensatory insertion or deletion (26a). Perusal of other paramyxovirus NPs in the data base indicates that this sort of problem is not unique.

The SEN andPIV1 NPs. To gain insight about the C-ter-minus of NP, we compared these regions in different pairs of paramyxoviruses. Comparison of very closely related pairs, such as parainfluenza virus 4 (PIV4) A and B or human and bovine PIV3 (hPIV3 and bPIV3, respectively) was uninfor-mative; there were too few differences here. Comparison of measles virus and canine distemper virus was also uninfor-mative, because there was

almost

no homology in this region, presumably because they were not sufficiently re-lated. However, comparison of

PIV1

and SEN, which are only slightly less related than the bovine and human strains of PIV3, was informative.

PIV1 NP is also 524 aa long and ends with the sequence GGI (19, 21). When aligned with SEN NP, the N-terminal 426 residues are 92% identical (conserved region 1 [CR1]), and the remaining C termini are relatively poorly conserved (47% identical). However, there are two blocks with stron-ger conservation within this tail (Fig. 1): a highly charged region in the middle (CR2, residues 463 to 488, 77% identi-cal) and a negatively charged region followed by a hydro-phobic one, representing the tip of the tail (CR3, residues 508 to 524, 65% identical). These two regions stand out because they are flanked by nonconserved regions.

We first examined the NP requirements for genome syn-thesis in vivo, where different NP plasmids were

cotrans-fected

(along,with

pGEM-P and pGEM-L) into cellsinfected with DIH4 . Genome replication here is dependent on NP,

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4360 CURRAN ET AL. A

DIH4 >

B I'll _v00

DIH4 > __

FIG. 2. DIH4 amplification in vivo with various NP proteins.

CV1 cell monolayersinfected with vTF7-3 were transfected with pGEM-P/C and pGEM-L plus different forms of NP as indicated above eachpanel. PIV1 referstoNP from hPIV1 andNPl-h and NPwt-freferto NPfrom the Harris and Fushimi strains of Sendai

virus, respectively. After 48 h of incubation, NC bands were isolated fromaCsCl gradient and phenol extracted,and the RNA

wasresolvedon a1.5% agarose-formaldehyde gel.The RNAwas

then transferredtonitrocellulosemembranes andprobedwitha(+) riboprobe transcribed from theplasmid pEX5'. The lanes marked

DIH4Uvarecontrols in which thehelper plasmidswereomitted and

indicate the level oftheinputDI in eachexperiment. Duplicatelanes represent separatebutparalleltransfections.

P, and L (3). After 48 h of incubation, NCswere isolated from these cells and their DI RNA levelswereestimatedby

Northern(RNA) blotting. Thebackground level dueto the

infectingor input genomeswas determined in each

experi-ment by omitting the helper plasmids (lanes DIH4Uv, Fig. 2). Each experiment also contained, as a reference, the

wild-typeNPgenefrom either the Harris(h)orthe Fushimi

(f) strain,which supportedgenomereplicationtovery sim-ilar levels(cf. singlelane NPwt-f withduplicatelanesNPwt-h, Fig. 2A).

Wefound that thePIV1NPgene(lanesPIV1)could in fact

replacethat of SEN and stillyield goodlevels ofreplication, i.e., 53% + 7% of NPWt (as determined in a

Phosphor-Imager). Thus, either the tail is unimportant here or only the conserved regions are important. Several C-terminal

truncations, NPPSt(or

NP1-518),

NpNco

(NP1-498),

andNpBal (NP'-456),werethen tested. Theseresultsareshown inFig.

2 andare summarized inFig. 1. Removing onlythe

hydro-phobic tipof the tail(residues519to524; NPPSt) produceda protein which was basically wild type in activity (Fig. 1).

NPNCo, which is missing the entire CR3, continued to

supportgenomereplication,butatathreefold reduced level. Residues 499 to 518 are therefore not essential for NP function inthis systembut contributetoactivity. NPBal,on the other hand, appearedtobetotally inactive,eventhough it was expressed at levels similar to NPWt as judged by

Western blotting (immunoblotting) (data not shown). Resi-dues 456 to 498, which include highly charged CR2, then

appeartobe essential forgenome replication.

We havecontrolled NP levels in all experiments, andwe report results

only

for those mutant

proteins

which were expressed to nearwild-type levels. Two constructs, even

though

they

weremade morethan once,

missing

the

C-ter-minal 98aa andone inparticular containing aframeshift in

codon 493 to emulate

PQQ-517 expressed proteins

which were toounstable for conclusionstobe drawn.

Differences between the in vivo and in vitro systems. In starkcontrasttoitsinabilitytosupportDIH4 RNAsynthesis

in vivo, NPBal

appeared

to be almostas active as NPWt in vitro (Fig. 3A). NPBaI, moreover, shared another property withNPWt,whichsuggestedthat itsactivityin vitrowasreal.

Intransfected cell extracts, P is foundas a

complex

not

onlywith Lbut also with unassembledNP(NP°), because

antibodies to one

protein

will cross-select the other from

coexpressed extracts (15). P-L is competent by itself for

transcription (6)

but

requires

P-NP°aswell for

replication,

the latter

presumably being

involved innascentchain

assem-bly. An

important

property of the in vitro system is that

transfected cellextracts areactiveonlywhen either all three

proteins

are

coexpressed [single

lanes

(NP

or NPBal+ P + L), Fig. 3A; the parentheses indicate which

proteins

were

coexpressed]

orPand Lare

coexpressed

inoneculture and P and NP are expressed in another and then combined

[duplicate

lanes

(P

+ NP or

NPBal)

and

(P

+

L)] (15).

In

particular,

NP is inactive unless

coexpressed

with P

[lanes

(P), (NP),

and

(P

+

L)],

even

though

its level in theextract

(like

those of P and

L)

is very

similar,

whether or not coexpression occurred

(Fig. 3B).

As shown in

Fig. 3A,

NPBal

was

just

as

dependent

on

coexpression

withPfor its

activityin vitroasNPWt.The

experiment

shown in

Fig.

3 has beencarriedoutthree times with

basically

the same

result;

NPBal appearstobe 40to 100% asactiveasNPWt in vitro.

The remarkable difference in

activity

ofNPBalin vivo and invitro mayreflect differences in what each system

actually

carriesout.

Multiple

rounds of

replication

occurin

vivo;

we

often find a 50-fold increase in DI genome levelsover the

input template by

Northern

blotting (e.g., Fig.

2A).

Because it is essential here to

completely

UV inactivate the

helper

genomes in the DI

stock,

relatively long

illuminations are carriedout to ensure

this,

with the consequence that

(i)

most of theDIgenomes

(probably

>90%)

arealso UV inactivated and cannot function as

templates,

but

(ii)

all will still be competentto

hybridize

the Northern blot

probe.

The50-fold increasewe see then more

likely

represents a much

larger

amplification

of the non-UV-inactivated

input.

In

vitro,

on

the other

hand, only

asmall amountof

highly

radiolabelled DI RNAis made froma

relatively large

amountof

template

(and

in a more limited

period

of

time),

andsoit is

possible

that only

synthesis

from the

input templates

is

being

mea-sured. If so, all thatis

being

measured in vitro may be the

ability

of NPto assemble thenascent chain

(and

drive the

polymerase

acrossthe

template).

Inthein vitro test, this NP neednotact, once

assembled,

as anessential componentof thedenovo-made

template.

If

NPBal

can support

replication only

from the

input

templates

carrying

NPWt,

genome

amplification

in vivo would be linear rather than

exponential,

and this very limited

synthesis might

notbevisible

by

blotting against

the

back-ground

of the total

input

DIs. It is therefore important to

specifically

measure the genomes made de novo in vivo.

Directly

radiolabelling

the transfected cultures is the best

approach,

but it is

impractical

here because of the small J. VIROL.

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FIG. 3. NPBa' supports DIH4 amplification in vitro, but only when coexpressed with P. RNA synthesis was carried outwith [32P]CTP in cytoplasmic extracts prepared from vTF7-3-infected A549 cells transfected with various combinations of pGEM plas-mids, asindicated above each panel. Parentheses indicateextracts

from a 5-cm petri dish in which the various constructs were

expressedorcoexpressed. Each petri dishwasusedtoprepare100 .1l ofextract,35 pl of whichwasaddedtoeach reaction. The final reaction volumewasadjustedto115 ,ul withanextractfrom cells infected only with vTF7-3, so that all reactions contained equal

amounts of cell extract. As negative controls, reactions were

performed in the absence of NP [lane (P+ L)]orin thepresenceof onlyP [lane (P)]. (A) Autoradiograph of the NC RNA from each

reaction thatwas isolatedfromaCsCl gradient and analyzedon a

1.5%agarose-formaldehyde gel. Duplicate lanes in the middle of the

figure represent separate but parallel transfections. (B) Equal

amountsof the various cellextracts(10 ,ul)weremonitored for NP, P,and L levelsby Western blotting withacombination of NP (W16),

P(1.180),and L monoclonalantibodies. NotethatNPBa'appears as adoublet band.

amount of synthesis expected with NPBal. We therefore

madeuse of another efficient system forgenome amplifica-tion recently developed, similar to that used above but in whichreplication isinitiated fromplasmid DNA rather than natural virus (DIH4Uv)infection (2a, 2b); therenowshould be no backgroundof input NCs. A(+)-DIH4 RNA is first transcribedbythe T7polymerase,andthis is thenassembled

andreplicated bytheNP,P,and Lhelperfunctions(lanes 3

and 4, Fig. 4A; the duplicate lanes represent separate transfections). When NP'W is replaced with NPBal, a very much smallerbut reproducible amountof (+)-DIH4 RNA is

found in the NC band regionof the CsCl gradient (lanes 5

and6, Fig. 4A). Autoradiographyis unsuited forquantitating signalsof suchunequal intensity, so aPhosphorlmagerwas also used, and these numbers are shown below the lanes. Note that there isnodetectablebackgroundin thisregion of the gradient in the control transfections (minus any NP plasmid; lanes 1 and 2, Fig. 4A), presumably because the

unencapsidatedT7 transcripts pellethere. Figure4Ashows

that NPBaI canin factencapsidate (+)-DIH4RNA in vivoto

a very small level, and this level could represent linear

amplification. When the same samples are examined for

(-)-DIH4 RNA,which should arise onlyif viralreplication

B: (+) probe

(-)DIH4 >

0 100 0.11

+11 + .11

FIG. 4. Genomereplication initiated from plasmid DNA. DIH4 genome replication was carried out as before, except that the

DIH4' infectionwassubstitutedby the addition of 5 p.gofplasmid

DNA which expresses (+)-DIH4 RNA via T7 polymerase (2b).

RNAs were recovered from the 1.31-g/ml region of the CsCl gradients and were probed on Northern blots for both (+)- and (-)-DIH4 RNAs (with riboprobes to the central region of these

RNAs).Thefigure showsan8-hexposureofthegels, whichwere

alsoquantitatedinaPhosphorImager,and thesenumbersareshown below the lanes. In eachcase,theaveragevalue of theNP' sample was set to 100. This value was three times as strong for the (+)-DIH4 RNA as that for the (-)-DIH4 RNA; however, this difference is dueatleast inpart tothe differentriboprobesused.

can occur, there isnowvirtuallyno PhosphorImager signal when NPWt isreplaced with NPBaJ (Fig. 4B), noris there a

PhosphorImager signalafterprolongedexposureof thegelto film (not shown). This experiment is consistent with the notion that NPBal iscapableofencapsidatingDIH4 RNA in

vivo, but the NPBal-RNA complex thus formed cannot be

used, or is used much less efficiently, for (-)-DIH4 RNA

synthesis.

Mapping boundaries ofregions requiredfor assemblyand

amplification. Because NPBal is still active forgenome

syn-thesis invitro,wetestedaseries of deletionmutants tomore closely define the regions required for this activity; the results are presented in Fig. 5. Deletion of residues 400 to 415,414to439,and 426to497, like NPBal(missingresidues 456to524),had littleor noeffectonactivityrelativetoNPWt. None of the C-terminal 125 residues of NP thenappeartobe essential foractivityinvitro,which ispresumablyrestricted

to nascent chainassembly. On the other hand, deletion of residues385to399 reducedactivity sevenfold,and deletions of short regionsbetween residues 34 and 384(mutantsB to R, Fig. 5)-including mutant N, which is missing only 7

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4362 CURRAN ET AL.

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-e - - - _ _ Y -_ _, >2

O6 = =L- = CECE =Ex= 7 aC V 3 0

01(/

0 = ID > :- a a CJ ) (1) aO 0.C t;

z X X < < mmm m m tn.<Z L Z [it

KB .1 .2

NP (F)

A B C D F G

K

Bglll-BgllI

L M

N

0

p

Q

R S T

0o

.0 u

II

a

m

. . . .3

.5 .6 .7 .8 .9 1 1.1 1.2 1.3 1.4 1.

.I

5

o~ 0

0 <

z

l1.6 1.7

- -

~~~~-

_m-

-!

-~~~~

-I-Eli

m~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

-- I - Eli mi

--I

li!m

--~l

- - Eli mi

-- I - Eli mi

--I - Eli m

I

-I=I

u

-EcoRV-Ncol Ball Ncol

SEN/bPI3 a2,,,z7777zz77/7777

AMINO ACIDS DELETED

1-33

34- 59 60-82 83-106 132- 152 153- 166 188-202

203-224 225-244

245-262

263-288 289- 295 296-318

319- 335

336-358 359-384

385- 399 400-415

414- 439 426-497

456-524 497- 524

[426-515]

GENOME SYNTHESIS

(%) 100

12

14

73

120 100 106

84

96

FIG. 5. The activity of various deleted NPs in vitro. A series of constructs containing deleted NP genes (reference 14 and Materials and Methods)were cotransfected into vTF7-3-infected A549 cells, and their cytoplasmic extracts were tested for theirability to support the synthesisofDIH4genome RNA in vitro (Materials and Methods). The regions deleted are indicated as openboxeswithin thegene, andtheir precisepositions are shown on the right. The hatched box of constructSEN/bPI3represents residues 426 to 515 of bPIV3. Thethin vertical lines on therightindicate the position of CR2. For genome synthesis, the numbers represent percent activity relativetoNPwt; a minussign indicatesnodetectable activity (<5%). NP levels in each extract were monitored by Western blotting andwere foundto besimilar tothose ofNP'W (not shown).

aa-led to NPswhich showed no activity above background

(i.e., <5%). Only mutant A, missing residues 1 to 33, showedsomeactivity above background. This suggests that CR1 is not only highly structured but that this structure depends on interactions which cover the entire region. Furthermore, changing the Leu-Val pair at either positions 229and 230 or 237 and 238 to Arg-Glu totally inactivated the protein in vivo, whereas changing Glu-489 to Gly or Pro reduced activity only two- and fourfold, respectively (not

shown).NPthen appearstobe composed ofaglobular head whichcantolerate almost no changes, with a C-terminal tail which forms a separate structure and which can accommo-date sequencechanges more easily. Only the globular head appears to be required for the RNA encapsidation, which naturally occurs concomitantly with genome synthesis.

Last, wenote thatthe phenotype ofNPBai isnot unique. Deleting residues 400 to 415 and 414 to 439, in particular, also eliminates activity in vivo but not in vitro. However, unlike

NPBal,

both ofthese mutants still contain the highly charged CR2 (residues 465 to 491). The residues between

position400 and CR2 are thus also important for activity in vivo. However, because residues 427 to 463 are very poorly conserved (Fig. 1), these residues may be important mostly

toposition the highly charged domain relative to the globular head,asopposed to containingsequences which themselves

are important for replication. Computer predictions of the

protein's secondary structure support this notion; residues 400 to 463 are predicted to contain several beta turns,

whereasCR2 itself is alpha helical. Interestingly, a chimeric

NP containing the entire CR1 of SEN (which is very well conserved with that of bPIV3) and the C-terminal tail of bPIV3 is also inactive in vivo (not shown) but is active in

vitro(Fig.5). However, its CR2 would be 10 residues closer

toCR1 than theNPWt CR2.

DISCUSSION

The central finding of this work is that thehypervariable C-terminal tail of NP is required for genome synthesis in vivo butnotin vitro. We have argued that in vitro, genome

synthesisis restrictedto thevastexcessoftemplates added

to the reaction, because diminishing their level 10-fold has

noeffect on netsynthesis(not shown). NP here then needs

onlytoassemble the nascent RNA during genomesynthesis.

Invivo,however,amuch smalleramountof DItemplates is usedtostarttheinfection, and genomesynthesis is detected

onlyafterallowing for multiple rounds of amplification. The

plasmid-expressed NPin the invivo system mustthen also beabletofunction aspartof thedenovo-made template. If so, theC-terminal tail ofNPisnotrequired fornascentchain assembly but presumably is required for the template to be functional. This is consistent with the finding that trypsin-treatedNCs, containingNPwhich ismissing the C-terminal 12 kDaof sequence(or roughly 100aa), aremorphologically

verysimilartountreated NCs(13, 24).Thebasicproperties

ofNPresponsible for the helical structure of the NC,orits formation, therefore donotdependontheC-terminal tail. In

addition, Buchholz et al. (la) have found an excellent correlation between the ability of these NP mutants to

assemble into NC-like structures(when expressed

indepen-dently)and theirabilitytosupport genomesynthesis in vitro. The tail accumulates changes more quickly during evolu-tion than the trypsin-resistant core. Yet two regions, one

highly charged in the middle of the tail and the other J. VIROL.

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[image:5.612.67.546.75.327.2]

negatively charged and then hydrophobic at the tip, are more conserved. Because deletion of this latter region (CR3) reduces genome amplification by only threefold, it presum-ably is conserved (in part) for functions other than RNA synthesis, e.g., for virion budding, in which it might interact with theamphipathic matrix protein (which has a net posi-tivecharge) (1). The 27-aa region, which is highlighted by sequence comparisons and appears essential for multiple roi nds of RNAsynthesis (CR2), contains 15 of 16 charged residues which aremostly conserved between SEN, PIV1, hPIV3, and bPIV3, with the negative charges distributed mostly at the N-terminal end (Fig. 1). This negatively charged end of CR2 has already been noted by Parks et al. (25a)as a commonregion of all paramyxovirus NPs. In the aboveviruses, this end also contains conserved Ser and Thr residues which could bephosphorylation sites, and so CR2 could beeven more negatively charged. However, a more detailed mutational analysis of CR2 will be required to detern ine whether these sequences aredirectly involved in multiple round genome synthesis.

What function th tailservesis unclear fromthiswork, but thereareseveral p ;sibilities of interest.

(i) The RNA within NCs is resistant toRNaseatanysalt concentration(13, 20), presumablybecause it isprotected by thesurrounding NP. During RNA synthesis, NPNCmaybe locally displaced from the template RNA so that the bases mayberead, and theC-terminaltail may berequiredfor this. Forexample, the tailmightsubstitute for thetemplate RNA boundby the N-terminal core,asuggestion previouslymade for the poorly conserved N-terminal half of the vesicular stomatitis virusphosphoprotein (17).

(ii)NPclearly playsa moreactive role in RNAsynthesis,

besides that ofsimply beingastructuralprotein.In the PolR

mutantsof vesicular stomatitis virus(26) and the Z strain of SEN (29), the polymerase reads through the leader-NP

junction athigh frequency, even in the absence of concur-rent assembly. In both viruses, this phenotype maps to

NPNC and not to the P-L polymerase in reconstitution

experiments (2c, 26). NPNC can then condition how the

polymeraserespondstojunctions,and the tailmightevenbe involved in this. The Z strain NP is also unique in that it

migrates anomalously fast during sodium dodecyl

sulfate-polyacrylamide gelelectrophoresis, andit maynotbe coin-cidental that this mobility determinant also maps to the

C-terminaltail

(unpublished observations).

(iii)Invivo,NP al (coexpressedwith P andL)appearsto

assemble RNAnonspecifically, because very abundant and

sharpNC bands formon CsClgradient centrifugation

inde-pendently of genome replication, and this does not occur

withNPWt.IntheCsClgradientsof theexperimentshown in

Fig. 4, for example, the visible

1.31-g/ml

bands from the

samples containing NPBal were severalfold stronger than those that contained

NPWt,

even though they contained

relativelyminuscule amountsof DIH4 RNA. In a separate

study,Buchholzet al. (la)have examined similarstructures with the electron microscope. Theywerefound to be very similarto naturally formed NCs and to contain RNA. The

C-terminal tail of NP may then also

play

a role in the

specificityof RNAassembly.

(iv)It isalsopossiblethat the tailsimplyregresentsasite

or sites atwhich Por Linteractswith NPN duringRNA

synthesis. We have begun to

investigate

functional NP-P

interactions by trying to rescue inactive chimeras between SEN and bPIV3 P by

complementation

with NP

chime-ras-sofar,without success.

ACKNOWLEDGMENTS

WethankJean-Baptiste MarqandCatherine Studer for excellent technicalassistance. We areparticularly gratefultoPhilippeCalain and Laurent Roux forprovidingtheimprovedDIH4vectorandfor

discussions.

This workwas supported by the Swiss National Science Fund. EMBOprovidedashort-termfellowshiptoH.H.

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