Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease.

0022-538X/87/041301-09$02.00/0

Copyright © 1987, American

Society

forMicrobiology

Processing of the Semliki

Forest

Virus

Structural

Polyprotein: Role

of

the

Capsid Protease

PAULMELANCONtAND HENRIK GAROFF*

European Molecular Biology Laboratory,Programof Cell Biology, 6900Heidelberg 1, FederalRepublicofGermany Received 21July 1986/Accepted1 December 1986

Theprotease activities responsible forthe cotranslational processing of the Semliki Forest virus structural polyprotein wereinvestigated by usinganin vitrotranscription-translation system. Three cleavages released theindividual chains from thenascentpolyprotein in theordercapsid,p62, 6K (a nonstructural peptide), and El.We showeddirectly that theproteaseactivityresponsibleforthe release of thecapsid protein resides in the capsid itself: by progressive truncation of the cDNA used for the SP6 transcription,weshowedthataprecursor containingasfewas38 residues ofthep62 protein leftattheCterminus of thecapsidwasstillveryefficiently cleaved in vitro.Wefurther tested the possibility thatserine-219 of the capsid is involved in autoproteolysisby site-directed in vitro mutagenesis. A changein the sequenceGly-Asp-Ser(219)-Gly, atetrapeptide conserved

among several animal serine proteases, to Gly-Asp-Arg-Ser-Thr was shown to completely abolish in vitro cleavage. Thissupportsthenotion that the capsid isaserineprotease. The role ofthecapsidproteasein the processing of the 6Kjunctionswastheninvestigated bytranslationsofahybridpolyprotein in which the capsid

andmostof the p62 sequences are replaced by those of the secretory protein Iysozyme. The cleavages and concomitant appearance ofthe6K peptide occurred efficiently and were shown to require the presence of membranes. This demonstrates thatthecapsidproteaseis notrequiredfor thosecleavages andsuggests that amembrane-associated hostproteaseisresponsible for the cleavage.

Semliki Forest virus (SFV) is a small, enveloped RNA virus that belongs to the genus Alphavirus of the family Togaviridae. It consists of a nucleocapsid containing the

single-stranded virus RNA complexed with a basic capsid protein,surroundedbyalipid bilayercontainingtwointegral glycosylated membrane proteins (E2 and El) and a small peripheral protein E3 (for areview, see reference 18). The translation of thestructural proteins of SFV is initiatedata single siteon asubgenomic 26S RNA. Threecotranslational

cleavage events release the capsid (33 kilodaltons [kDa]), p62 (62 kDa; a precursor of the E3 and E2 proteins), a

nonstructural 6K peptide (60 amino acids [aal long), and finally the El protein (50 kDa).

The first cleavage event occurs between the tryptophan (aa 267) and serine (aa 268) of the polyprotein and releases thecapsidprotein from the polysome. The newly exposed N terminus of the p62 protein is then involved in initiating chain translocation across the endoplasmic reticulum (ER)

membrane. Chain translocation continues until it is arrested atthetransmembrane segmentof thep62protein but is then reinitiated to allow translocation of the El protein. The signal for this second translocationeventresides within the 6K peptide, the nonstructural peptide presentbetween the p62 and El proteins (36). It remains unknown whetherthe twocleavage events that are responsible for the release of thep62, 6K, and Elproteins occurbefore, during, or after the reinitiation oftranslocation. A final cleavage converts theP62glycoproteintoE2 andE3. This lastcleavage ismost probably a late Golgi event occurring en route to the cell surface.

The chymotrypsinlike protease responsible for the first cleavage (between C and p62) is probably virus encoded. The strongest evidence comes from the in vitro study of

*Correspondingauthor.

tPresent address:DepartmentofBiochemistry,Stanford

Univer-sitySchool ofMedicine, Stanford, CA94305.

Aliperti and Schlesinger (4). Using the fact that the transla-tion of26S RNA of SFV and the related Sindbis virus in cell-free systems gives riseto rapid and efficient release of the capsid protein (7, 9, 11, 12, 19, 21, 41), these authors have shown (i) that the appearance of the capsid protein

could be inhibited by the addition of aa analogs in the cell-free translation system and (ii) that almost complete cleavage of the accumulated precursor could be observed

after a chase with cold and normal aa. Theseresults dem-onstratethatde novosynthesis (from the viral RNA) of the

protease isrequired and that the proteasecan actintrans. Several observations suggest that the viral protease is located near the N terminus of the polyprotein. First, in

synchronized in vitro translations, the capsid protein is liberatedvery soonafter it issynthesized (8, 19). Second, the

analysis of the sequence of the cDNAs of temperature-sensitive(ts)mutantsdefectiveincleavage(and their

respec-tive revertants) has shown that the criticalaa substitutions

were located in the capsid protein (25). Moreover, the sequence surrounding serine-219 in SFV (serine-215 in Sindbis virus) shows homology with that around the serine present atthe active centerof several serine proteases (6). For these reasons, and the fact that one ofthe mutations discussed above mapsclose to serine-215, itwasproposed that the protease activity responsible for the capsid-p62 cleavageresides within thecapsidproteinand that itactsas a serineprotease(25).

Incontrast, thecleavagebetween the6K and Elproteins is most probably catalyzed by the host protease signal peptidase.We haverecentlylocated the translocationsignal forthe Elprotein to the last 26 residues of the6Kpeptide (36). This region contains a stretch of hydrophobic aa residues andhasadistribution of small and basicresiduesat thecleavage sitecharacteristic ofsignal peptidase cleavage sites. Much less is known about the cleavage between the p62 and 6k proteins. If that cleavage occurs in the cyto-plasm, the protease involved is most probably virus en-1301

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coded,

since no host

cytoplasmic

protease with the

appro-priate

specificity

has yet been described. The

possibility

therefore exists that the

putative capsid

protease

plays

some role in the

p62-6K

cleavage.

In thework

reported here,

weusedtheclonedcDNAfor the SFV 26S RNAto

study

the involvementof

capsid

in the

processing

ofthe

structural

polypeptide. By truncating

from

the 3' end the cDNA used as a

template

for in vitro

transcription-translation

experiments,

we showed

directly

thattheprotease

activity

is restricted tothe

capsid region.

Second,

we introduced

by

in vitro

mutagenesis

two aa

changes

at the

putative

active serine-219 and showed that

this

completely

abolishes

capsid cleavage.

Thissupportsthe notion that the

capsid

is a

serine

protease.

Third,

using

a

hybrid protein

in which the

capsid protein

and the luminal

ectodomain of

p62

was substituted with chicken oviduct

lysozyme

(a

secretory

protein),

we demonstrated that the

capsid protein

was not

responsible

for the

cleavages

on

either side of the6K

peptide.

MATERIALS AND METHODS

Materials. All

restriction endonucleases,

as well as calf intestine

phosphatase,

T4 DNA

polymerase,

Escherichia coliDNA

polymerase

1

(Klenow fragment),

and SP6

poly-merase, wereobtained from

Boehringer

GmbH, Mannheim,

Federal

Republic

of

Germany. Mung

bean nuclease

(fast

protein liquid

chromatography grade)

was

purchased

from

Pharmacia,

Freiburg,

Federal

Republic

of

Germany,

BAL31

was

purchased

fromBethesda Research

Laboratories, Inc.,

Gaithersburg, Md.,

RNasin was

purchased

from

Promega

Biotech, Madison,

Wis.,

and

proteinase

K was

purchased

from E. Merck

AG,

Darmstadt,

Federal

Republic

of

Ger-many. T4 DNA

ligase

was agenerous

gift

ofE. Winkler.The

ribonucleotides

(equine)

and

spermidine

usedforRNA

syn-thesis werefrom

Sigma

Chemical

Co.,

St.

Louis,

Mo. The

deoxy-

and

dideoxyribonucleotides

usedforDNA

sequenc-ing

and thecap

analog 7mGpppG

werefrom Pharmacia. The DNA linkers

(Sall

octamer)

andthe SP6 promoter

primer

were obtained from

Boehringer.

Ribonuclease

A,

phenyl-methylsulfonyl fluoride, iodoacetamide, lysozyme,

creatine

phosphate,

and

creatine

phosphokinase

were from

Sigma.

The rabbit

reticulocyte

lysate, L-[35S]methionine,

L-[35S]cysteine,

and

low-range

molecular

weight

(MW)

mark-erswere

purchased

from Amersham

Corp., Braunschweig,

Federal

Republic

of

Germany.

The

middle-range

MW

mark-ers and

En3Hance

were obtained from New

England

Nu-clear

Corp., Dreieich,

Federal

Republic

of

Germany.

Seph-adex G-75and

Sephacryl

S-300werefromPharmacia.

Low-melting-point

agarose was

purchased

from Bethesda

Re-search Laboratories. Salt-washed

dog

pancreas

rough

microsomes

(50

A280

units/ml)

were

provided by

B.

Dobberstein. Nikkolwas a

gift

fromD.

Meyer.

General DNAmethods. For

rapid screening, plasmid

DNA was

prepared by

the

boiling

methodofHolmes andQuigley

(29),

as described

previously (43).

Large

quantities

of

plas-mid were

prepared by

a modification of the alkali-sodium

dodecyl

sulfate method of

Birnboim

and Doly (5), as

de-scribed

previously (36).

DNA

fragments

wereisolated over

low-melting-point

agarose. Restriction endonucleases and

DNA-modifying

enzymes were used as specified by the

manufacturer. All molecular biological manipulations were

performed by

standard

protocols

(35). The preparation of

competentbacteria

(E.

coli

HB101)

and theirtransformation were as described

by

Hanahan (26). The sequencing of double-stranded

pGEM plasmids

by using the SP6 primer wasdone

essentially

asdescribedbyChen and Seeburg (10).

Construction of the vectors. Late simian virus 40vectors for in vivoexpression were constructedasfollows.The SFV cDNA for these constructions was obtained from

plasmid

pSV-S-SFV(37) as aHindlIl fragment. The cohesive ends of the isolatedfragmentwerefilledin,andBamHIlinkerswere added, (Fig. 1A). Thisfragmentwasinserted at the BamHI

site ofvectorpLl (originally described as pAll-SV-L-2by

Gruss et al. [23]) to produce pLl-SFV. This vector was modifiedby substitutingapieceofpBR327for thelarge XbaI pBR322 fragment originallypresent,thereby removing

sev-eralNaeI sites and apoison sequencethat interferes in cis

with simian virus 40 replication (34). Plasmid pBR327 was cut with EcoRI and AvaI and treated with S1 nuclease to

destroy the EcoRI andAvaIsites (Fig. 1). Afterrepairwith

DNA polymerase, XbaI linkers were added, and the small

fragmentwaspropagatedas aplasmid.Thisplasmidwas cut

withXbaI, treated with phosphatase, andsubstituted for the pBR322 fragment between the XbaI sites of pLl-SFV to

produce pL2-SFV.

The modification of the capsid-encoding region was

ac-complished with the exonuclease BAL31. Aplasmidwith an

out-of-frame insertion ofalinkeratthe site ofmodification was first constructed (Fig. 1). Plasmid pL2-SFV was cut

extensively with NaeI untilmostof the DNA was linearized.

Theendswerefilledin, the linear fragmentwasisolatedover agarose, and a

Sall

linker (octamer)was added. Aplasmid containing the linker attheproperlocation, pL2-SFVN/S,

was chosen for further work. SalI linker insertion was necessary to overcome thevery inefficientdigestion atthe

NaeI site ofinterest.

Plasmid pL2-SFV N/S was cut to completion with Sall and treatedbriefly withBAL31inabuffercontaining 0.5M

NaCl at 30°C. After inactivation of the nuclease, the ends wererepaired withDNApolymerase, and Sall linkerswere

added. Ampicillin-resistant transformants were first

screened

by

restriction forthe presence ofplasmids with a

Sall linker. Positiveplasmidswerethen screenedby

trans-fection of COS cells for the

synthesis

of full-length El

precursor, as described below. Since the starting plasmid could not

synthesize

El protein as a result of the out-of-frame insertion of the

linker,

any plasmid in which the translation reading frame had been restored following the

gentle

BAL31 treatmentmusthave sufferedamodification in thesequenceof interest.Approximately one-fourth of the plasmids tested containedalinker thatwasin frame with the

restofthecodingsequence.Thesewerefurther analyzed by restrictionwithAvaItodeterminethe extentofthedeletion. PlasmidpL2-SFV

6/,

showingaminimalmodification, was

chosen for further work. The 216-base-pair AvaI fragment

was subcloned at the AvaI site of plasmid pGEM 1 and

sequenced by using the SP6 promoterprimer.

Transcription vectors for in vitro expression. The SFV insertsofplasmids pL2-SFV and pL2-SFV 6/7were isolated as BamHIfragments andinserted at theBamHI site of the SP6 vectorpGEM 1(PromegaBiotech)(Fig. 1B).

The cDNA for chicken lysozyme was obtained from

plasmid pSV-2-Lys (32). The HindIII lysozyme insert was subcloned atthe HindIII site ofthepolylinker of pGEM 1. Thehybridgeneforaprotein containing almost the entire sequence oflysozyme fused to the C-terminal half of the SFVpolyprotein was constructedby a three-fragment

liga-tion. The lysozyme portion wasisolated as a HindIII-NaeI

fragment,and that for SFV was isolated as a ScaI-BamHI

fragment (Fig. 1B). The vector was reconstructed with a

HindIII-BamHI fragment containing almost the entire

pGEM 1 plasmid.

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BamHl Xba1 _A

/ ~Xbal Hid

BapHl id SFV )

BamHl fSl-in

Xba Bl BamHn s - 3

EcoRl

pL1-SFV ,L

C ) / ~~~~~~~~~~Aval

Xbal aem1 Aval_EcoR?

Stnuclease

Xba1 ~~~~~~Fill-mn XbaI Xba?Inkers

sBamHl

Xbal Nael cI~ Xbal

X7;XbaI

pL2-SFV

Xbal i J

BamHB

Naae Ba&/i Xbal Sal linkers No

BamHrl pL2

Xb 1 > L S 11 \ ( / \\ ~~~~~~~~Xbal

pL2-SFV

N/S

Xbal

SalI Ba131 Salinkers

Xbal BamHl

pL2-SFV

Xbal 6/7

BamHl

BamHr

Hind3 Hind3 IHind3

|PSVLy/Hind3

P/,nd3 Nael ,Nael

~/ Hind3 Nje1 \BBamHl

Hind3 Na.?

Bar/l 7d3

cI

Hind3 Hind3

BamHl 4

_S Nael

\ Hind3//

Peul

[image:3.612.152.472.66.396.2]

BamHl

FIG. 1. Outline oftheplasmid constructions for in vivo (A) and in vitro (B)expression studies. Thin lines indicate pBR-derived DNA

sequences,thick lines indicatesequencesfrom simian virus40,andboxed regionsrepresentSFVcDNAorchickenoviductlysozymecDNA

(hatched areas). The largestboxesrepresentcodingregionswithin the cDNA sequences.All circlesare drawnto scale(forcomparison, pSV-S-SFV is 7,603 base pairs long). For detailsseeMaterials and Methods.

Transfection of COS cells.Plasmid DNA for the screening of in-frame pL2-SFV N/S mutants was prepared by the

boiling method. The DNA (2 pig) wastransfected on COS cells grown on cover slides (5 mmby 5 mm) by using the

DEAE-dextranmethod described by Cutleretal. (13). The

appearanceof the SFV El proteinwasdetected by indirect

immunofluorescence in Triton X-100-solubilized cells by a modification of themethod described by Timmetal. (43). As

anegative control, COS cellsweretransfected withplasmid

pL2orunmodifiedpL2SFV N/S. In thosecases, no or very

few cells were positive for El. In about one-fourth of the transfections with the modified PL2-SFV N/S DNAs, 5 to 10% ofthe cells in the monolayer showed bright reticular staining. Thepolyclonal rabbit anti-El antibodywas a gen-erous gift of D. Louvard and G. Warren. The

rhodamine-conjugated anti-rabbit secondary antibody was a generous

gift of Thomas Kreis.

Transcription and translation. CappedmRNAwas

synthe-sized in vitro from the pGEM vectors derivatives by using thebacteriophage SP6 polymerase, asdescribedpreviously (30, 37).LinearizedplasmidDNA(0.5 pug )wastranscribed at40°Cwith3to4U of SP6polymerase inafinal volume of 10 pd containing 0.5 mM each ATP, UTP,andCTP,0.1 mM GTP, 0.25 mM 7mGpppG, 10 U of RNase inhibitor, 2 mM spermidine, 100 ,ug of bovineserumalbuminperml,40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.4), 6mM magnesiumacetate, and 10

mMdithiothreitol. Aftera 10-min incubation, anadditional

100pmol of GTPwasadded, and the reactionwascontinued

for a further 30 min. A 25-,Il rabbit reticulocyte cell-free translation assay mixture contained 2 pul of transcription

mixtureand 50%commerciallysate andwasadjustedtothe following final concentrations: 100mM potassium acetate, 1.2 mMmagnesiumacetate,0.4 mMspermidine, 40 ,uM each 19 aa minus methionine (or cysteine), and 60 ,Ci of L-[35S]methionine(orL-[35S]cysteinewhenindicated). Someof the translations were carried out with dog pancreas salt-washed membranes present at aconcentration of 2.0 A280

units/ml. The membranes were treated onice with 25 mM

ethylene glycol-bis(,-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (pH 7.5) and with 0.005% Nikoll (Nikko, Tokyo, Japan) priorto addition. Translations were

carriedoutat30°C for60min.

Protease assay. Proteinase K digestion of the translation products was carried out at0°C for 30or60 min at afinal concentration of 0.25mgofproteaseperml in thepresence

orabsenceof 1% Triton X-100. Proteolysis wasstopped by the additionoffreshly dissolvedphenylmethylsulfonyl fluo-ride(20mg/mlinisopropanol)toafinalconcentration of1 to 2 mg/ml. After incubation for 5 min at 0°C, 2 volumes of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample bufferwasadded and thesampleswereimmediately heatedto95°Cfor 5 min.

Electrophoresis. Samples from confluent monolayers of

Sca? BamHl

IScal

BPvul

BamHl

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BHK-21 cells infected with SFV

(wild

type or

ts3)

were

prepared

as described

by

Green et al.

(22).

The infected

monolayers

were labeled5.5 h

postinfection

with 50

,uCi

of

L-[35S]methionine

for4 h

(ts3)

orwith

L-[35S]cysteine

for10

min

(wild type).

Some of the infected cells were

pretreated

with4 ,gof

tunicamycin

per mlfor2.5 hand labeledfor4h

in the presence of

tunicamycin.

Cells were

lysed

with hot

sodium

dodecyl

sulfate

sample

buffer and

passed

several

times

through

a Hamilton

syringe

to break down

chromo-somal DNA. All

protein samples

were heatedto

95°C

for5 minin thepresenceof dithiothreitol and then

alkylated

with

iodoacetamide.

Electrophoresis

of cell-free translation

prod-uctsof8 and10%

polyacrylamide

slab

gels

and

subsequent

processing

for

fluorography

were

essentially

as described

previously (13). Electrophoresis

on22%

polyacrylamide

slab

gels

containing

6 Murea was done as described

by

Swank

and Munkres

(42).

Middle-range

MW markers were

phos-phorylase

b

(92,500),

albumin

(69,000),

ovalbumin

(46,000),

carbonic

anhydrase (30,000),

and

lactoglobulin

A

(18,370).

Low-range

MWmarkerswere carbonic

anhydrase (30,000),

trypsin

inhibitor

(21,500), cytochrome

c

(12,500), aprotinin

(6,500),

andinsulin B chain

(3,400).

RESULTS

Cleavage

of the

capsid protein

in a cell-free

transcription-translation system. The

processing

ofthe

polyprotein

was studied

by

using

a cell-free translation system

programmed

with RNA transcribed in vitro. The construction of the

plasmids

used as

template

for RNA

synthesis

is outlinedin

Fig.

1 and is described in more detail in Materials and

Methods.

Templates

for RNA

encoding

the

complete

poly-protein

were

prepared by

linearization ofthe

pGSFV

plas-mids withanenzyme,

PvuII,

thatcuts verynearthe 3' end of the

4-kilobase-long

SFV cDNA in the vector sequence.

Translation-competent

RNA was

produced by transcription

with the

phage

SP6

polymerase

in the presence ofthecap

analog 7mGpppG.

Under such

conditions,

the RNA

pro-duced has the size

expected

fora

transcript

ofa4-kilobase cDNA

(data

not

shown).

A rabbit

reticulocyte lysate

pro-grammed

with the

wild-type

RNA

(labeled

SFV in lanes 2

and

5)

produced large quantities

ofa

protein

ofapparent MW

33,000

(Fig.

2).

This

protein

has almostthesame

migration

in

polyacrylamide gel

electrophoresis

asdid

capsid produced

in BHK cells infected with the SFV ts3 virus

(lane

4). (Note

that the ts-3 virus infection was carriedout at

semipermis-siveconditions

[37°C]

toensuretheaccumulation of boththe

polyprotein

precursor

[MW, 130,000]

and the structural

protein products.)

The

slight

differencein

migration

ratemay

be duetothe mutationin

ts3,

which is knowntoresideinthe

capsid

protein,

orto some

posttranslational

covalent modi-fication of thesame which occursin

vivo,

orboth

(33).

We

conclude that the

capsid protein

is very

efficiently

synthe-sized and cleaved from the nascent chain in our in vitro

system. This is in agreement with

previous

translations

performed

with26S RNAisolatedfrominfectedcells (7, 9,

11, 12, 19,

21, 41).

Someofour

wild-type

translations were

more efficientand allowed the

synthesis

ofa distinct

[image:4.612.347.526.364.624.2]

high-MW

protein species (Fig.

2,

lane

2)

inadditiontothecapsid

protein.

This 97-kDa

product

must correspond to the

C-terminal

portion

ofthe

polyprotein (containing

thep62,6K,

and El

sequences)

that is left after

cleavage

ofthe capsid

protein

(19, 33).

No material

corresponding

tothe

full-length

polyprotein

observed in ts3-infected cells

(pl30)

(lane 4) accumulates in the

wild-type

RNA translations (lane 2). Note thatlanes1and 6

(see

also

Fig.

3,lanes2and4) show

analyses of the

polyprotein

with a mutated

capsid

protein

thatwill be described below.

Protease activity resides within the capsid protein. The protease

activity

encoded

by

the

wild-type

RNA was

mapped

by progressively truncating

from the 3' end the cDNA

template

used for the

synthesis

of the RNA. The RNA prepared from templates cleaved with the enzymes NcoI andXhoI encode proteins that nowcontain only 159 and 38 residues of p62 attached to the

capsid

protein,

respectively(Fig. 3,top).Ifthese

proteins

didnotcontainan active protease we would expect the accumulation of pre-cursorsof 50 and 37kDa, respectively.The

fluorogram

(Fig.

3, bottom)shows that the 33-kDacapsidband isproducedin thelysates programmedwith bothtruncated RNAs(lanes 1 and3). The C-terminal fragments released

by

the

cleavage

are toosmalltobe observedon ourgels.Sincenodetectable amountof 50- and 37-kDa precursor molecules

accumulates,

thecleavageof thecapsidappearsinbothcases toberather efficient. This demonstrates

directly

that the protease

activ-ityresides within thecapsid protein, since it isvery

unlikely

that the 38-residuefragmentofp62has suchan

activity.

Changes at serine-219 destroy the proteaseactivity. As first notedby Boegeetal. (6),the sequencearoundserine-219 of thecapsidof SFV(serine-215in Sindbisvirus)has

homology

with a tetrapeptide sequence conserved around the active

residue in several animal serine proteases. Inaddition,theaa substitutionresponsiblefor thecleavagephenotype ofsome

~> ~>

A

L/ 'iV

ri>

B r

p130--92k

-92k --b9k

-46k

- -30k

1 2 3

-69k

E1,

2[I

-46k

capsid

-30k

4 5 6

FIG. 2. Cell-free translations of RNA transcribed from full-length wild-typeandmutantSFVcDNA. Thein vitro transcription-translation ofplasmids pGSFV (labeledSFVinlanes2and5) and pGSFV6/7(labeledSFV6/7 in lanes1and6) linearized with PvuII

wascarriedout asdescribedinMaterialsandMethods. The trans-lation products from two different experiments were separated eitheron a10%gel(A)oron an8%gel (B). Lysateobtained from BHKcells infectedat 37°CwithSFV-ts3 was included inthe gel shown inlane4.Standard molecularweightmarkersarealsoshown (describedindetail in Materials andMethods).Thefigurerepresents

afluorogramof thegel.

J. VIROL.

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ts mustants ofSindbis virus (ts2 and ts5) has been shown to map atresidue218, near the putative active serine-215. We

wished to further test the hypothesis that the capsid is a serine protease by changing the sequence at the serine-219

residueanddetermining the cleavage phenotype in vitro with

complete and truncated precursors. Taking advantage of a

NaeIsite that overlapsthecodon forglycine-220,thecDNA

was mutagenized in vitro by using the enzyme BAL 31 and

linker addition,asdescribed in Materials and Methods. The nature of the changes introduced in one of the mutated

cDNAsequences (SFV6/7)wasobtained by DNA

sequenc-ing (data not shown). The deduced wild-type and mutant

protein sequences are shown in Fig. 4: a new arginine

residue was introduced on the N side of serine-219, and

glycine-220 was changed to threonine. Transcription-translation experiments with the full-length and truncated

mutatedtemplates were carried out as described above for

the wild-type one. The results with the full-length template

(Fig. 2, lanes 1 and 6) clearly show that no capsid can be

produced from the mutated cDNA. Instead, one observes

the accumulation of a high-MW band, absent from the

wild-type translations. Thisnew band comigrated with p130

produced inBHK cells infected with ts3 SFV (lane 4) and most probably corresponds to the full-length unprocessed

polyprotein.

(Note that many other medium-sized products

areseeninlanes 1 and 6. Theseprobablyrepresentvarious runoff products formed during translation ofthis very long mRNAinvitro.) Weconclude thatthe limitedchanges that we haveintroduced clearly destroy the protease activity. As

Xhol

38

Nco I z

159

SFV

267 488 60 438

capsid p62 6k El

X ' X

s0 m0

o o ( )< 2

-69k

-46k

capsid -0 do

-30k

1 2 3 4 5

FIG. 3. Analysis ofthe autoprotease activity ofthe capsid by truncation of the wild-type and mutant cDNA templates. Top, Diagramsof theexpectedtranslationproductsof the RNAprepared

fromthefull-lengthcDNA(SFV)and from cDNAs truncated with therestrictionenzymesNcoI and XhoI. The sizes of the individual

capsid p62andElproteinsand those of theexpected p62fragments

arealso indicated. Transcription-translation wascarriedoutasfor

Fig.2. Theproductswereseparatedon a10%gel.Thefluorogramof

thatgel ispresentedinthe lower part of thefigure.Thenatureofthe

templateis indicated above each lane.

w.t.

capsid mutant

C () p62 6k El

r6IyvAsPSer GlTy-(219)

c x p62 6k El

FIG. 4. DiagramoftheSFV polyprotein,indicating the sequence of theconserved and modified tetrapeptidethought to beinvolvedin theautoproteaseactivity of the capsid protein.

expected, translations withthetruncated mutated templates

(XhoI 6/7andNcoI 6/7) did not produce capsideither, but,

instead,theyproducedlargerproteins that have the mobili-tiesexpected fortheunprocessedtruncated precursors(Fig.

3, lanes 2and4). These resultsfurther demonstrate thatthe

cleavages observed withthe wild-type templates were

spe-cific and required the presence of a wild-type capsid se-quence. We believe that the mutation does not act by changing the structure of the cleavage site, since (i) the

changes were introduced 48 residues away fromthe siteof action ofthe protease and (ii) Aliperti and Schlesinger (4)

have shown that the introduction of aa analogs in the

substrate didnotpreventcleavageby the wild-typeprotease. Ourresultssupport thenotionthat theregion around

serine-219isimportant forproteolysisand that thecapsidis aserine

protease. Capsidprotease isnotinvolved incleavageofthe

p62-6Kjunction.

Thein vitro cleavageofthe6Kjunctionsandreleaseofthe

p62andElproteins has been observed onlyin the presence

ofmicrosomes (7, 19). Asdiscussed above, weexpect that the

6K-El

cleavage is catalyzed by signal peptidase atthe luminal surfaceof theERmembrane(36).This doesnotrule

outthepossibility that the other cleavagebetweenp62 and

El occursinthe cytoplasm, however.The latterprocessing

eventmayrequiremembranesfortheattachmentof the p62

protein andpropertopologyofthe 6Kpeptide relativetothe ER membrane. Toaddressthe possible involvement ofthe

capsid proteasein thecleavage ofthe 6Kjunctions,thegene

for a hybrid protein was constructed that was missingthe

capsid sequence but maintained the regions thought to be

important for correct topogenesis of the 6K peptide. The

construction ofa hybridgene with the cDNAs fora secre-toryprotein, chicken lysozyme, and the SFVpolyprotein is outlinedin

Fig.

1 anddescribed in moredetail inMaterials

and Methods. The resulting hybrid protein, Lys-El, is representedin Fig. 5. Allbutthe C-terminmal2 residuesof

thelysozymesequence arepresent,includingthe 18-residue cleavable signal sequence. This was substituted for the

capsid and most of the p62

protein.

The

hybrid

protein

retains the transmembrane segment and tail of the p62 protein in addition to the total 6K and El sequences. We expect that during translation ofthat

hybrid

protein

in the presenceofmembranes, translocationwillbeinitiated

by

the

lysozyme

signal

sequence, then arrested

by

the transmem-brane region of the p62 protein, and

finally

reinitiated to allow the translocation of theEl

protein, leaving

thetailof

p62(30 aa)and

possibly

somepart of the 6K

peptide

exposed

in thecytoplasm. Upon

translocation,

the

signal

sequenceof lysozyme, as well as the 6K-El

junction,

should be effi-ciently cleaved

by

signal

peptidase.

The release of the 6K

peptide will be

dependent

onwhether

capsid

isinvolved in thep62-6Kcleavage.Incase

capsid

isnot

involved,

the 6K

peptide should be

generated

in addition to a

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1306 MELANCON AND GAROFF

lysozyme

18 129

127

66

I

11s) (

267 433 1 24 1 31 60 412') 24 V2

[image:6.612.61.295.73.167.2]

Capsid p62 6Kpeptide E1

FIG. 5. Diagram of the hybridprotein constructed tostudythe involvement of the capsid protease in the processing of the 6K peptide. The intact proteins encoded by the chicken lysozyme cDNA and the SFV26S cDNAareshownassingle lines above and below, respectively. The vertical bars represent proteasecleavage sites, and the wavy lines indicate the approximate positionof the lipidbilayer. The portions of thetwoproteins present in the hybrid areshown in the middle as boxes connectedbyavertical line. The size of theprotein domains bound by the bars and wavy lines are also given.

bound form of the lysozyme molecule (MW, 18,5000) and

finallyEl. On the otherhand, werecapsid requiredfor this cleavage event, the 6K peptide would remain as a C-terminal

extension ofthetransmembranelysozyme.Theapproximate MWofsuchahybrid is 26,000.

Theresults of the cell-free transcription-translation with thehybridgene areshowninFig. 6 and 7. To detect the 6K

peptide (Fig. 6), translations were performed with

L-:E: vr

!:

L

30k -SM 21.5k-12.5k

[35S]cysteine (the 6K peptide has five cysteines and onlyone methionine) and the products were separated on a high-resolution gel containing urea. Under suchconditions, one can detect the appearance of a small band (lane 4) that

comigrates with the 6-kDa band produced in BHK cells infected with SFV (lane 2). This bandcanariseonly if both 6Kjunctionswerecleavedin vitro. Note that theprocessing of the 6K junctions occurs only in the presence of microsomes (compare lanes 3 and 4). Figure 7 shows the analysis ofa L-[35S]methionine translation on a 10% poly-acrylamide gel, onwhich the larger products canbebetter resolved. In the absence ofmicrosomes, amajor product of the size expected for the unprocessed Lys-El hybrid (65 kDa) accumulated (Fig. 7, lane 2). A minor abortive product migratingat50.5 kDawasalso present. When thetranslation wascarried out in the presence of microsomes (lane 3), less Lys-El precursor was seen and two new major bands appeared: one migratingat18.5kDa and anotherone migrat-ingat50 kDa (just below the abortive product of 50.5 kDa). The bandat18.5 kDamostprobably represents membrane-bound lysozyme which arises from (i) translocation of lysozyme with concomitantcleavage of the signal sequence, (ii) arrest of translocation at the transmembrane region of p62, and (iii) correct cleavage of the p62-6K junction. Note that no band with anapparent MW of26,000(corresponding

y ys-El

69k --y

4w _d_o

46k

-30k

-

6.5k-- - 6k peptide _18k

-3.3k- A r,

in vitro -M +M

1 2 3 4

FIG. 6. In vitro generation of the 6K peptide. Transcription-translation ofthelysozyme-El hybrid protein was carried out in the presence(lane 4) and absence (lane 3) of membranes, as described in Materials and Methods, with

L-[35S]cysteine

forlabeling. The prod-ucts were separated on a 22% gel containing 6 M urea. The fluorogramis presented. Lysate from BHK cells infected with SFV was also included (lane 2). The MW markers (low range) are described in Materials and Methods.

MM M M

p p p TX

1 3 4 5 6

FIG. 7. Cell-free translation of thelysozyme-Elhybrid protein. Transcription-translation was carried out as described in Materials and Methods.Translations were done in the absence (lane 2) and presence(lanes3to6) of salt-washed microsomes (M). Some of the translationproducts were treated with exogenous proteinase K (P) for30(lanes4and6)or60(lane 5) min intheabsence(lanes4and 5)orpresence(lane 6) of Triton X-100 as described in Materials and Methods.

IS

.:

4

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[image:6.612.390.481.337.649.2] [image:6.612.114.250.385.645.2]

to a membrane-bound lysozyme with the 6K peptide at-tached) is observed. The above interpretation is supported by the protease challenge experiments shown in lanes 4 and 5. The membrane-bound lysozyme is resistant to the exogenous protease K and is shifted down by about 2 kDa. This is exactly the shift expected if translocation has oc-curred, the anchor of p62 spans the membrane, and the tail of p62 remains exposed to the cytoplasm (13, 20). As expected, the patternwas not affected by doubling the time of digestion from 30 to 60 min (compare lanes 4 and 5). No protection from the protease was observed when the diges-tion was carried out in the presence of Triton X-100 for 30

min(lane 6). The other major band, the one with an apparent MW of 50,000 (lane 2), most probably represents glycosyl-atedEl. It is protected from the added protease (lanes 4 and 5) and comigrates with glycosylatedElobtained from SFV-infected BHK cells which have been pulse-labeled with

L-[35S]methionine(data not shown) (36). The weak protected band migrating in front of glycosylated Elat approximately 48 kDa has been observed before in vitro translations and represents translocated unglycosylated El chains (36). We conclude that the cleavage of the 6K junctions does not require capsid but, as discussed below, does require a membrane-associated host protease. We further conclude that the transmembrane region of p62 can anchor the luminal domain of a foreign protein. Similar observations have been made with the anchors of other transmembrane proteins (24, 46).

DISCUSSION

We have established an in vitro transcription-translation system for studying the cotranslational processing of the SFV structural polyprotein. Since the RNA used for the translations is produced in vitro with the SP6 polymerase, the cDNA templates can be modified at will and the effect on the processing can be conveniently assessed in a rabbit reticulocyte cell-free system. The in vitro system was used first to study the protease responsible for the release of the capsid protein. We demonstrated directly that the activity resides within the structural protein capsid by using a combination of two approaches. (i) The cDNA used for transcription was truncated with restriction enzymes, and we showed that a capsid precursor containing only 38 additional residues from the N terminus of the p62 protein was very efficiently cleaved in vitro. (ii) The cDNA was mutagenized at the site of the putative active serine-219

residue, and this was shown to completely abolish the cleavage of the capsid protein. Since theprocessingrequires de novo synthesis (4) and since it is very unlikely that the 38-residue fragment of p62 has any activity, we conclude that the capsid is an autoprotease, responsible for its own release from the polysome.

The evidence to suggest that the SFVprotease isaserine protease has been recently reviewed by Hahn etal. (25). In the work described in this paper, we directed our mutationto the serine-219 thought to be part of the catalytic triad of the serine protease activity in the capsid and found that the mutation completely abolishedcleavage. Sincethemutation

is far removed fromthe siteofcleavage (48residues away), it is unlikely to act by changing the nature ofthe substrate. Our results therefore support the notionthat the capsid is a serine protease. Previous attempts to obtain in vitro direct evidence by using specific inhibitors such as tolylsulfonyl

phenylalanyl chloromethyl ketone have failed (4) and were not repeated inthis study. A definitive proofofthecatalytic

[image:7.612.322.555.68.149.2]

a b c d

FIG. 8. Summary of the various cotranslational protease cleav-ages which are required to generate the SFV structural proteins. Details are given inthe text.

role ofserine-219 and the mechanism ofthe

capsid

protease in general requires the use of a series of mutations with

well-defined single-residue substitutions around each of the residues at theproposed active site.

The residues postulated to be involved in the serine protease activity of thecapsidmap to theC-terminalpartof

the molecule (25). This part, in contrast to the N-terminal one, ishighlyconserved (14, 39).

Interestingly,

this homol-ogy has recently been extended to

the

coat

proteins

of picornaviruses (S. Fuller and P. Argos, unpublished

data),

the crystal structure of some of which has

recently

been solved (28,40).Onthebasis of this

finding

(and

thefactthat the alphavirus nucleocapsid is arranged with the same

(tri-angulationnumber,T = 3) icosahedral symmetryasthoseof

the picornaviruses; S. Fuller andJ.

Dubochet,

unpublished

data), it waspredicted that the C-terminalpartof the

capsid

polypeptide is folded into a eight-strand

P-barrel

structure typical of the shell domains found in all the available high-resolution structures oficosahedral viruses

(1, 27,

28,

40). Theregionsthoughttobe involved in the

serine

protease

activity ofthealphavirus

capsid

coincide on one side ofthe

predicted 3-barrel. The positioning of the protease active site within a structural element of the virus

clearly

differs

from the arrangement in picornaviruses. The

picornavirus

polyprotein is composed ofa structural and anonstructural

region. Almost all the cleavages,

giving

rise to both struc-tural and nonstrucstruc-tural

proteins,

are

catalyzed

by

two proteases which are located in the nonstructural part of the

molecule (fordiscussion, see reference

44).

Thecapsid of SFV appears tobeinvolved in

only

a

single

proteolytic cleavage event, which releases the C sequence from thatofp62.The fact thatweobserved correct process-ing at the 6K peptide in the Lys-El

hybrid

protein

demon-strates clearly that the

capsid

protease is not involved in these other cleavage events of the SFV structural

polypro-tein. At present, we favor a model in which both 6K

cleavagesarecatalyzed

by

the host protease

signal

peptidase

in the ER lumen (see also reference

39).

This model is

supported bythe facts that

(i)

cleavages

occurin vitro

only

in the presence of microsomal membranes and

(ii)

both

cleavagesites arepreceded

by

a

signal

peptide-like

sequence (45), i.e., a hydrophobic stretch of aa

plus

a

particular

distribution around the

cleavage

site. These features are present at the C-terminal of

p62

and 6Kin all

species

of the

genus Alphavirus in which these sequences have been deter-mined (SFV [18], Sindbis virus

[39],

and Ross River virus [14]). Figure 8 summarizes

schematically

our model for the way in which we think that the various structural

proteins

are formed from the

polyprotein.

The

drawing

first illustrates the

autoproteolysis

ofthe

capsid

protein

occurring

in cison the nascent chain. A

cis-acting

capsid

protease must be

involved toaccountfor the first

capsid

protein.

The

experi-ments ofAliperti and

Schlesinger

(4)

suggest that

capsid

can f

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http://jvi.asm.org/

alsoactintrans. The

newly exposed

Nterminusofp62acts as a

signal

sequence andinitiatesthetranslocation ofthep62

protein (stage

"a" in the

drawing).

The

signal

sequence

(represented by

an empty

rectangle)

is not cleaved but

becomes

glycosylated

andtransferredtothe lumen(stage b).

Translocation of the

p62 protein

is arrested by the trans-membrane segment,

represented by

asolid

rectangle

(stage

c).

A

possible

arrangement of the

p62

tail and 6K

peptide

allowing cleavage by signal peptidase

for both 6K

junctions

is

depicted

instages d andeof the

drawing.

Thehydrophobic

region

at the C terminus ofp62

(residues

395 to

428,

open

rectangle)

could act as a

signal

sequence that initiates the translocation ofthe 6K

peptide through

the ER membrane. The 6K

peptide

isshown anchored in the membrane viaan

internal, 15-residue-long apolar region (residues

22 to

36,

solid

rectangle),

with the N terminus in the lumen. It has been shown that a 15-residue

hydrophobic region

is

long

enough

to serve as a membrane anchor

(2, 3,

13,

15,

16).

Note that such stretches of

hydrophobic

residues are also present in the middle ofthe 6K

region

of Sindbis virusand RossRiver virus

(14,

39).

The

hydrophobic

Cterminus ofthe 6K

peptide (residues

45to

58,

open

rectangle),

asmentioned

above,

acts asthe

signal

fortranslocationof the El

protein

(36). Upon cleavage by signal peptidase,

the tailof

p62

must

flip

back on the

cytoplasmic side,

since it is known to be

available to antitail antibodies

microinjected

in the

cyto-plasm (13;

L.

Roman,

unpublished observations).

The same is

thought

to

happen

with the El translocation

signal

(stage

f).

This kind of

flip-back

has been

reported

for the

signal

sequence of

preprolactin

after it had been fused to the C terminus of the

alpha-globin protein (38). Finally,

stage f shows the El

protein fully

translocated andanchored

by

its

transmembrane segment

(solid rectangle).

We

hope

to use our in vitro

transcription-transla-tion/translocationsystemtotestthe model of 6K

processing

and

topology.

The

temporal relationship

between thetwo6K

cleavages

and thetranslocationof the El

protein

canalso be

studied with this system.

ACKNOWLEDGMENTS

WearegratefultoT. Kreis and G. Warrenforgenerousgiftsof antibodies. ThanksarealsoduetoB.Dobbersteinforprovidingsalt washedmicrosomes.Furthermore,we are mostgratefultoS. Fuller forthecommunication ofunpublishedresultsandfor criticalreading of the manuscript. We thank L. Roman and D. Cutlerfor useful discussions in theearlypartof this work.

P. MelanconwastherecipientofaNorthAtlanticTreaty Organ-izationpostdoctoral fellowship, obtainedthrough theNational

Sci-ence and Engineering Research Council of Canada, and of a short-termEuropean MolecularBiology Laboratory fellowship.

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