Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.

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JOURNAL OF VIROLOGY, Mar. 1992,p. 1610-1621 Vol. 66,No.3 0022-538X/92/031610-12$02.00/0

Identification

and

Characterization

of

an

Extracellular

Envelope

Glycoprotein

Affecting Vaccinia Virus

Egress

STEPHEN A. DUNCANAND GEOFFREY L. SMITH*

Sir William Dunn SchoolofPathology, University ofOxford, South Parks Road,

Oxford

OX]

3RE,

United

Kingdom

Received 25 October1991/Accepted4December 1991

Sequence analysis of thevaccinia virus strain Western Reserve genome revealed the presence ofanopen reading frame (ORF), SalLAR,which has thepotentialtoencode atransmembrane

glycoprotein

withhomology to

C-type

animallectins(G.L.Smith,Y. S.Chan,andS. T.Howard, J. Gen. Virol.72:1349-1376, 1991).Here weshowthat the SalL4Rgeneistranscribed lateduring infection fromaTAAATG motif at thebeginningof the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified threeglycoprotein species ofMr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is presentinpurified extracellularenveloped virusparticles butnotinintracellularnakedvirus(INV).A mutant viruswasconstructed by placinga copy ofthe SalL4RORFdownstream ofan

isopropyl-13-D-thiogalactopy-ranoside(IPTG)-inducible vacciniaviruspromoter withinthethymidinekinase locus andsubsequentlydeleting theendogenous SalL4R gene.Thegrowthkineticsof thisvirus demonstrated thatSalL4Rwasnonessential for theproduction of infectious INVbut was required for virus dissemination. Consistent with thisfinding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed thatwithout SalL4Rexpression, the inability of the virus tospread is due to a lackof envelopment of INVvirionsby Golgi-derived membrane, amorphogeniceventrequired forvirus egress. Vacciniavirus, the prototype of the genusOrthopoxvirus,

replicates in thecytoplasm of infected cells and contains a

large double-stranded DNA genome with the capacity to encode approximately200proteins (14). Anincreasing num-ber of these proteins have been identified, and their func-tions in the virus life cycle have been elucidated, thus contributingto agreaterunderstandingofpoxvirus biology.

These functions range from roles in DNA replication and transcriptional regulation to the inhibition of host defense mechanisms (reviewed in references 27 and 53). However, the genes controllingvirus morphogenesis and the mecha-nism mediating the spread anddissemination ofpoxviruses

in tissue culture and throughout the infected host remain

poorlydefined.

Morphogenesisofvaccinia virus results in theproduction

oftwoinfectious forms of virusparticles: intracellularnaked virus (INV) and extracellular enveloped virus (EEV). The

majority of virus particles produced are INV, and

conse-quently this is the most intensely studied form of vaccinia virus. A small proportion of INV becomes wrapped in a double layer of Golgi-derived membrane (20, 26) which contains several virus proteins. Enveloped particles then

migratetothe cell surface, where the outer membrane fuses with the cellplasma membrane, releasingEEVfromthecell. EEVisresponsible for the enhanced dissemination of prog-eny virions in cellculture and within the infected host and is therefore fundamentaltothepathogenesis of a vaccinia virus infection (2, 3, 32).

EEVcontains 10 virus proteins which are not present in INV, 9 of which are glycosylated (30, 31), and which give EEV distinct immunological and biological properties. The genes encodingonly two of these proteins, a 37-kDa (37K)

acylated protein (18) and the 86K hemagglutinin (46), have been identified. Other components of EEV are a group of

*Corresponding author.

fiveglycoproteinsin the range of 20Kto 23K and

glycopro-teins of42K, 110K, and 210K(31).

The molecular mechanism by which EEV leaves the infected cell ispoorly understood. The process is blocked by

glycosylation inhibitors, e.g., 2-deoxy-D-glucose and glu-cosamine (35), andby N1-isonicotinoyl-N2-3-methyl-4-chlo-robenzoylhydrazine(IMCBH),which prevents the

envelop-mentof INVbyGolgi-derivedmembrane(33). Mutations in theacylated 37Kprotein(F13L)of the EEVouterenvelope

confer resistanceto IMCBH and thusimplicatethisprotein in EEV egress fromthe cell (44). Thevaccinia virus fusion protein 14K (A27L), which is present on both INV and infected cell membranes, is also required for INV envelop-ment (42). An intact cytoskeleton is also reported to be necessary for EEV formation, because cytochalasin D, which inhibits microfilament formation, prevents EEV re-lease from the cell surface (34). Although glycosylation inhibitors inhibit EEVrelease, the roles of specific vaccinia virusglycoproteinsinvirus egress and dissemination are not understood (35, 56). In this report,we present the identifi-cation andtranscriptional analysis ofavaccinia virus gene

encoding a group of 22K to 24K glycoproteins that are

associated with EEV particles and which are required for virusegress.

MATERIALSANDMETHODS

Cells and viruses. CV-1, B-SC-1, BHK-21, RK-13 and human TK-143 cells were grown in Glasgow's modified Eagle's medium (GMEM) containing10%fetalbovine serum

(FBS). Vaccinia virus strain Western Reserve (WR) was propagated in either CV-1 or BHK-21 cells as previously described (23), and the IHD-J strain was grown in RK-13 cells. Recombinant viruses expressing

Escherichia

coli

xan-thine-guanine phosphoribosyltransferase (Ecogpt) were se-lected andpurifiedonfresh monolayers of CV-1 cells under an agarose overlay containing GMEM, 2.5% FBS, 6 ,ug of 1610

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VACCINIA VIRUS EEV GLYCOPROTEIN 1611

mycophenolicacid (MPA) per ml, 250 ,ug of xanthine per ml,

and 15 ,ug ofhypoxanthine perml as described previously (12). Thymidine kinase-negative (TK-)recombinant viruses wereselected and then plaque purified three times in fresh

monolayers of human TK-143 cellsin thepresenceof 25 ,ug of 5-bromodeoxyuridineper ml. Recombinant virus vSAD9 wasgrown in the presence of 5 mM isopropyl-,B-D-thiogalac-topyranoside (IPTG).

Purified INV stocks of WR or IHD-J virus were prepared byinfecting either BHK-21 or RK-13 cells, respectively, at 0.1 PFU per cell for3 days. Cells were broken by Dounce homogenization, the nuclei were removed bycentrifugation at 800 rpm, and virus was pelleted through a 36% (wt/vol)

sucrose cushion as previously described (23). For some experiments, this semipure virus was further purified by sedimentation in a 15 to 40% (wt/vol) continuous sucrose gradient (23). IHD-J EEV was purified from RK-13 cells infected as described above. Culture medium was clarified

by centrifugation at 2,000 rpm for 10 min, and supernatant

virus was pelleted(13,500 rpm, 4°C, 60min)and banded in a

continuous sucrosegradient asdescribed above.

Growth curves. Viruses used for growth curves were

grown in BHK-21 cells and semipurified in parallel as

described above. B-SC-1 monolayers were infected with

either 10or 0.001 PFU per cell in the presence or absence of 5 mM IPTG. After 90 min, the monolayers were washed threetimes with 2 ml of phosphate-buffered saline (PBS)and

then overlayed with GMEM containing 2.5% FBS, with or without 5 mM IPTG. Intracellular virus was measured by collecting infected cells by centrifugation (2,000 rpm, 10

min),

resuspending the cell pellet in 1 ml of GMEM, lysing

the cells by three freeze-thaw cycles, sonicating the cell

lysates,and titrating the virus on freshduplicate monolayers ofB-SC-1 cells.

Construction of plasmids. (i) Plasmids used for constructing recombinant viruses. A 1.9-kb SpeI fragment containingthe

SalL4Rgene was excised from the vacciniavirus(WR) SalI

Lfragment and cloned intoXbaI-cutpUC13 to formplasmid pSAD2. To inactivate the SalL4R open reading frame

(ORF), a 414-bp fragment (81%) of this ORF was removed

by digestion of pSAD2 with NaeIfollowedbypartial

diges-tion with ScaI. This fragment was replaced by a 2.1-kb

EcoRI fragment, rendered blunt ended by treatment with

Klenow enzyme, containing the vaccinia virus 7.5K pro-moter driving the Ecogpt gene derived from plasmid pGpt

07/14 (4). The derivative plasmid was called pSAD8.

A copy of the SalL4R ORF flanked by BamHI sites and with an additional NdeI site upstream was made by poly-merase chain reaction (PCR) using pSAD2 DNA template.

The oligonucleotides used, 5'-CCCGGATCCATATGAAA

TCGCTTAATA and

5'-CCCGGATCCTCACTTGTAGAAT

TTT, represent the 5' (positive strand) and 3' (negative

strand) of the ORF, respectively, and include BamHI and NdeI sites (underlined). The 519-bp PCR

product

was di-gested with BamHI, cloned into BamHI-cut

pUC13,

and

sequenced to check thefidelity of the Taq polymerase. The

ORF was then excised as a BamHI fragment from this

plasmid, pSAD5, andinserted intoBamHI site downstream

of theinducible p4b vaccinia viruspromoter within

plasmid

pPR35(41) to formplasmid pSAD15. This

plasmid

wasused to insertan

IPTG-inducible

form of the SalL4R intothe TK locus ofthevirus genome.Toproduceaprobe forthe

region

of theSalL4R ORFdeleted inpSAD8andfree from

possible

contamination with other vaccinia virus

DNA,

a

269-bp

AccI-ScaI

fragment was isolated from pSAD2, rendered

blunt endedbytreatmentwithKlenow enzyme,and cloned intoHincII-cut pUC119toform pSAD23.

(ii) Plasmids for expression of SaIL4R in bacteria. To construct aplasmid expressing a TrpE-SalL4R fusion

pro-tein, a 362-bp AccI fragment that contained the

carboxyl-terminal 61% of the ORF was isolated from pSAD5. This fragmentwas made flush ended with Klenow enzyme and ligated into the end-repaired EcoRI site of the

expression

vectorpATH3(22) toformpSAD24. The reestablishment of thepATH EcoRI siteas aconsequenceof theinsertionwas

consistent with theSaIL4R

fragment being

in frame with the TrpE ORF.

Expression of a SalL4R fusion protein in E. coli. E. coli HB101 cells

containing plasmid pSAD24

were grown until

mid-log

phase in 10 ml of LB broth

containing

50 ,ug of ampicillinperml.The cellswerethen

pelleted, resuspended

in a total volume of 100 ml of M9

medium-ampicillin,

and shaken at 37°C for 60 min. The inducer

indoleacrylic

acid

was added to a final concentration of 20

pug/ml,

and the culture was incubated with

shaking

for 4 hat

37°C

before

being

left

overnight

at

4°C.

Bacteria were

pelleted,

resus-pended in 50mM Tris-HCI(pH

7.5)-S5

mM EDTA-3 mg of

lysozyme

perml, and leftonice for2 h. Cellswere

lysed

by

theaddition of0.35 MNaCI-0.75% Nonidet P-40and incu-bated on ice for 30 min. DNA was sheared

by

sonication before

proteins

presentininclusion bodieswerecollected

by

centrifugation

at

10,000

rpmfor10min. Pelletswerewashed in 1 M

NaCl

containing

10mMTris(pH

7.5)

and then in 10 mMTris(pH 7.5)priorto

resuspension

in1mlof10 mMTris (pH 7.5).

Proteins,

denatured

by boiling

for 5 minwith an

equal volume of2x

protein sample

buffer

(125

mM

Tris-HCI

[pH

6.8], 4%

sodium

dodecyl

sulfate

[SDS], 40%

glycerol,

1 M

,B-mercaptoethanol,

0.002%

bromophenol

blue),

were

resolvedona

preparative

12%

polyacrylamide-SDS

gel.

The gelwasstained with0.05% Coomassie brilliant

blue,

and the

TrpE-SaIL4R

fusion

protein

was excised andelectroeluted

by

using

aRennerGmbH

protein

electroeluterapparatus.

Production and affinity

purification

of antisera. New

Zealand White rabbits were inoculated with 750

jig

of

TrpE-SalL4R

protein

in Freund's

complete

adjuvant.

At

2,

4, and 8 weeks after the initial

inoculation,

rabbits were

boosted with

750-,g aliquots

offusion

protein

in Freund's

incomplete

adjuvant,

andserum

samples

taken2weeks after boosts. An

immunoglobulin (Ig)

fraction of anti-SalL4R

serum was obtained

by

ammonium sulfate

precipitation

followed

by

DEAE

affinity

purification,

using

methods

pre-viously described (16).

Anti-SalL4R-specific

antibody

was

further

purified

from the

Ig

fraction

by

affinity

chromatogra-phyon

TrpE-Sepharose

4Band

TrpE-SalL4R-Sepharose

4B

columns. Protein-linked

Sepharose

columns were

prepared

by

using

10 mg of either

(i)

protein lysate

from

HB101

cells

containing plasmid pATH3

andinduced toexpress

TrpE

or

(ii)

purified

TrpE-SalL4R

fusion

protein.

The

Ig

fraction from 20 ml of rabbit anti-SalL4R serum was twice

passed

through

the

TrpE-Sepharose

4B

column,

and the nonad-sorbed eluatewascollected and

applied

tothe

TrpE-SalL4R-Sepharose

column. Boundanti-SalL4R

antibody

waseluted

with 100 mM

diethylamine (pH

11.5)

andneutralized with 0.1 M

NaH2PO4

to

pH

7.5.Theanti-SalL4R

Ig

wasconcentrated and

equilibrated

in PBS in an Amicon concentrator.

Ig

specific

for SalL4R was demonstrated

by

its

ability

to

recognize

the

TrpE-SalL4R

fusion but not

TrpE

protein

in

Western immunoblots

(data

not

shown).

Western

blotting.

B-SC-1

cells

(106)

infected with the

appropriate

viruses at 25 PFU per cell were maintained for

24 hin thepresenceorabsenceof5 mMIPTG and/or1,ugof

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1612 DUNCAN AND SMITH

tunicamycinper ml. The cells were washed inPBS, scraped

from the dish, collected by

centrifugation,

resuspended in 0.1 ml ofPBS, and lysed by addition of0.1 mlof2x protein

sample

buffer. After

shearing

of the DNA by brief

sonica-tion,

the

proteins

were resolved on a 15%

polyacrylamide-SDS

gel

and

electrophoretically

transferredtonitrocellulose membranesas described

previously

(16). These membranes

were incubated with rabbit anti-SalL4R, diluted 1/50 in Tris-buffered saline at pH 8.0

(TBS)

containing

5% nonfat driedmilk

(BLOTTO)

for2hat roomtemperature, and then washed in TBS. The immune complexes were detected by incubation with 1 p,g of alkaline

phosphatase-conjugated

donkey

anti-rabbit Igper ml in5% BLOTTO-TBS and then

withbromochloroindolylphosphate-nitroblue tetrazoliumas

described

previously

(16).

Immunoprecipitation. B-SC-1 cells

(106)

infected withthe

virusat25 PFUpercellwere incubated for15 min with 100

,uCi of [35S]methionine in methionine-free GEM at 6 h

postinfection (hpi).

When

required,

1 ,ug of

tunicamycin

per ml was included throughout. After labeling, medium was

removed, and cells were washed twice with PBS and then

harvested

directly

or chased with medium supplemented with 2 mM methionine forafurther 1, 2, or 3 h. Cells were

lysed

in 1.5 ml of

phospholysis

buffer (PLB) (11) (0.01 M

NaPO4

[pH 7.4], 0.1 M

NaCl,

1% Triton X-100, 0.1% SDS,

0.5%

sodium deoxycholate) before centrifugation in a

mi-crofuge

at

4°C.

Then 5 lI of antiserum wasadded to0.5 ml

ofcellextractin1mlofPLB. After incubation of ice for4h, immune complexes were reacted for 90 min at 4°C with

protein

A-Sepharose, collected by centrifugation, and

washed twice with PLB. Bound proteins were eluted in

protein

sample buffer, boiled for 3 min, resolved on a 15%

polyacrylamide-SDS gel,

andidentifiedby autoradiography

using preflashed

film.

Immunogold labeling of virus particles. Carbon-coated

copper400-mesh

grids

(a

gift

fromA. C.

Minson, University

of

Cambridge)

werefloatedon4 x 107PFUofIHD-J INV or EEV

purified

virus

particles

in 96-well tissue culture dishes. Unless otherwise

indicated,

the

grids

werewashedwithPBS and TBS for 2 min and then in 50% ethanol for 30 s.

Virus-coated grids were then incubated for 15 min in TBG

(TBS

[pH 8.2], 0.1% bovine serum albumin [BSA;fraction

V],

1%

gelatin)

before being transferred to wells containing either

anti-SalL4R,

anunrelatedrabbitserumdiluted1/50in

TBS containing 1% BSA, or affinity-purified

anti-SalL4R-specific

Ig diluted 1/5 in the same buffer. After 50 min of

incubation at room temperature, virion-coated grids were

washedfor 10 min in TBG, and bound Ig was detected by

incubation for 30 min in colloidal gold-conjugated goat

anti-rabbitIg diluted1/10 inTBScontaining 1%BSA. Grids werewashedfor5 minwith TBG andthen TBS, after which

proteins

were fixed in TBS containing 2% glutaraldehyde.

Virus particles were finally negatively stained with 2%

uranyl

acetate.

Southern blotanalysis. Vaccinia virusDNAwasextracted from virus cores as described previously (10) and digested

withKpnI. Thefragmentswere resolved on a 0.8%agarose

gel

before

being

transferred onto nitrocellulose (24). Blots were probed with (i) a 269-bp

KpnI-HindIII

fragment iso-lated from pSAD23 representing DNA deleted from the

SalL4R gene in pSAD8, (ii) a

KpnI-EcoRI

fragment from

plasmid pGpt

07/14 (4) containing 1.6 kb of sequence from the Ecogpt cassette but lacking the vaccinia virus 7.5K promoter, and (iii) a TK gene fragment produced by PCR upon a TK DNA template by using oligonucleotides

5'-CCCAAGCTTTTAATTAGACGAGTTAGA

and 5'-GGGA

AGCTTCTATCTCGGTTTCCTCAC, which represent,

re-spectively, sequences within upstream

(positive

strand)

and

downstream (negative strand) regions ofthe vaccinia virus (WR) TK gene. Probeswere labeled with[a-32P]ATP, using arandom-primed DNA labeling kit(Boehringer

Mannheim)

as instructed bythe manufacturer. Hybridization and wash-ing were performed as previously described (50).

S1 nuclease protection analysis. Early and late vaccinia virus RNA was prepared from CV-1 cells infected in the

presence or absence of cycloheximide as previously de-scribed (49). A 32P-labeled DNAprobe designed to

identify

transcripts initiatingnear the 5'endof SalL4Rwasmade

by

digesting pSAD2 with AccI, purifying a 3.6-kb DNA

frag-ment, dephosphorylating this fragment with calf intestinal alkaline phosphatase, and labeling the 5' ends with [_y-32P]ATP by using polynucleotide kinase. ThisDNA was digested with BcIl, and a 431-bp fragment was purified.

DNA-RNA hybrids were formed by precipitating 5 x

103

cpm of labeled probe with 10 jig of early or late vaccinia

RNA or with 10 ,ug ofyeast tRNAwith isopropanol andthen redissolving the precipitate in 30 ,ul ofhybridization buffer [40 mMpiperazine-N,N'-bis(2-ethanesulfonic acid)

(PIPES;

pH 6.4), 1 mM EDTA, 0.4 M NaCl, 80% formamide] and incubating it overnightat 37°C. Single-stranded nucleic acid

was digested with 1,000 U ofS1 nuclease in 0.28 M

NaCl-0.05 M sodium acetate-4.5 mM ZnSO4 for 60 min at25°C.

Protected fragments were separated on6% polyacrylamide sequencinggelsanddetected by autoradiography.

RESULTS

The sequence and deduced ORFs within theSall L, F, G,

and I fragments ofthe vaccinia virus (WR) genome have

been reported elsewhere (48). To identify genespotentially

encoding glycoproteins, ORFs were screened for the

pres-ence of signal sequences (25, 54) and the amino acid motif N-X-S/T, which acts as the attachment site for

asparagine-linked carbohydrate. Within this 42 kb of sequence a number of ORFs encoding putative glycoproteins were identified,

and the genomicposition of one of these, SalL4R, is shown

(Fig. 1A).This 507-bp ORF ispredicted to encode a primary translation product of 168 amino acids with a molecular

weight of 19,539 (48). The hydropathy profile ofSalL4R (Fig. 1B) reveals the presence of a 22-amino-acid hydrophobic sequence near the N terminus of the predicted protein. This

mightfunction as a signal and anchor sequence for a

mem-brane-bound protein with a class II topology, such as the influenza virus neuraminidase (13) and the human transferrin receptor (45). The predicted protein sequence contains a single putative N-linked glycosylation site toward the car-boxyl-terminalendof the protein (48).

A comparison of the deduced amino acid sequence of

SalL4R with sequences in the SWISSPROT data base (ver-sion 15), by using the program FASTA (37) revealed

homol-ogy to a number of C-type animal lectins (9), to a groupof fowlpox virus (FPV) proteins (52),and to the vaccinia virus

SalF2R protein (48). An alignment of SalL4R with these proteins (Fig. 2) shows a number of conserved residues predicted to contribute to a lectin fold; however, some conserved residues of other C-type animal lectins (28) are absent from SalL4R, suggesting that although SalL4R and theaforementionedproteins arise from a common ancestry, SalL4R might not function as a lectin. The similarity of

SalL4R to the FPV proteins and vaccinia virus SalF2R suggests the existence of a new family of poxvirus lectin-like proteins.

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VACCINIA VIRUS EEV GLYCOPROTEIN 1613 A

C NMK F E PO I G L J H D A B

U1 I I I I I I I

.,

I I

,

. ,

l

, ,

,

/

F IGL9I| I~~~~/

SalL4R

(507bp)

B

phob

3.0

-1.5

-1.

0

-1.5S

-3.0

phil

I1

JIG

.11

,

II.

0 34 68 101 135 169

FIG. 1. (A) TheHindIIImapof the vaccinia virus (WR) genome showing the position of the 507-bpSalL4R ORF ( ) within theregion sequenced by Smithetal.(48) (1111 ). The positions of the SalI L, F, G, and I fragments are expanded below. (B) Hydropathy profile for the predicted 168-amino-acid SalL4R protein. The x axis indicates the amino acid number starting from the N terminus.

SalL4R is transcribed late in the virus life cycle. The presence of the late transcriptional initiation sequence

TAAATG (15, 43, 55) atthe 5' of the SalL4R ORF and an

early terminalsignalTTTTTNT (57) withinthecoding region

implies

that SalL4R is transcribed late during infection. Consistent with this

view,

Northern (RNA) blot analysis using a

SaIL4R

strand-specific probe identified

heteroge-neously sizedRNAslate ininfectionbut noearly transcripts

from this region (data not shown).

Si

nuclease protection wasused toconfirm this finding andtomapthe start site(s)

of the late SalL4R transcripts. A

5'-32P-labeled

probe

(Ma-terials and Methods and Fig.

3B)

was partially protected from

Si

nuclease digestion when hybridized to late viral

RNAbutnottoearly viralRNA orcontroltRNA. Thesize of the protected fragment, determined by comparison with

an M13

sequencing

ladder, mappedthe late transcriptional

startsitetothe TAAATGmotifatthe 5' endofSalL4R (Fig. 3A).

SalL4R

isrequired for normal plaque formation. Tostudy the roleoftheSalL4Rgene

product

inthe virus lifecycle,a

virus lackingthe SalL4R ORF wasrequired. We attempted

toconstructsuchavirusby replacingtheSalL4RORFwith

theEcogptgene linkedto thevacciniavirus 7.5Kpromoter

and selecting MPA-resistant viruses(4, 12), a strategy

pre-viously used to construct a virus lacking the DNA ligase gene (21). However, after transfection of WR-infected cells with pSAD8(aplasmid inwhich the

SaIL4R

gene isreplaced by Ecogpt) and analysis of the genome structures of 30

MPA-resistant recombinants, no virus lacking the SalL4R

gene wasidentified(data notshown), implying thatSalL4R

wasrequired for vaccinia virus growthin tissue culture. As an alternative approach to study the essentiality and

functionof

SalL4R,

aviruswasconstructedthatexpressed this gene from an IPTG-inducible late vaccinia virus

pro-moter (41) within the TK locus and had the endogenous SalL4R gene deleted. A similar approach was previously used to demonstrate that the vaccinia virus 14K protein is

required for virusegress(42). WR-infected cells were

trans-fected with

pSAD15 (Materials

and

Methods),

and a

TK-recombinant virus, vSAD7, containing an IPTG-inducible

copy oftheSalL4Rgene within the TK

locus,

was selected and plaque

purified

in the presence of

bromodeoxyuridine.

The SalL4Rgene wasthendeleted from its native locus

by

transfecting

plasmid pSAD8

(see

above)

into vSAD7-in-fected CV-1 cells. Recombinants

containing

Ecogpt were

selected and plaque

purified

in the presence of both MPA and IPTG. The

resulting

virus, vSAD9,

contained a

single

IPTG-inducible SalL4Rgene.

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[image:4.612.149.490.80.449.2]

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c L

4*kNIFQIR*4FGKGTKdIARY*I3DMEGQ

V WIG

IISPE2rFI

IHASHTLAV

IIHQEi'FQTNAGVA

Lectin C_WYQ_YIIIDAQ TVHPGA) RTVSIS

Sa1F2R Tr1LLSDR2EGR1sALPsr. ~TPJlJS S.IRR

jjjjij]ITNIKMST NVTKRRLRP DTRH XStF)

EI C,KIW,EFDNIISENKLDSWNLGGGN I I

C

Ej=M 4FSEEcKNSLAVE*

MDMDGH

NU

'DNIETIkJM

NTNSGiI

SKE3 'Y}KD 'RYGKGS) IT SKD' RYKGPGNI

Consensus F

CD23

lIjr

AFP I W

AcB Lectin YIW

W SalF2R TE4X VV SalL4R

FPV Bam2 INFSI

FPV Bamll II FPV Bam8

ISDGTP1

ISNGEATE Y W

9iI8PGEPT SRSQGEI ;FSTKPD D VLAAC

E¶SNNPN NW ENQI

BAKNATIQG TKKRKYI INNDKDIDISKLTNFKQLNSTTDAEJ(IYKS

;LYY EDGVNDICLLFDTSNIIE

:NNCN F;NIVGCDIC TIERFYl

C WD C C

flnM

GSGE

RKLGAW4E3RLATCTPPASE

MQIMTAAAD AS HKS4CNMTF MTVTKNjIIIIrGCPPCGI

TPKLH*ZITI

GKLVKST QSVI§nFYK

iRTIC VKAJYTHWYTEYMR

FIG. 2. Alignment of the amino acid sequence of the SalL4RORFwith sequences of relatedpoxvirus proteinsand with sequences of members of theC-type animal lectinfamily.Aligned proteinsequencesarefrom humanIgEFcERII(CD23),Megabalanusrosa(searaven) anti-freezepolypeptide (AFP), acorn barnacle lectin(AcBLectin),vaccinia virusSalF2R(VVSalF2R),vaccinia virusSalL4R(VV SalL4R), FPVBamHIORF 2(FPVBam2), FPV BamHI ORF 8(FPVBam8),and FPVBamHI ORF11(FPV Bamll).Residues above the top lineare thosedescribedasbeingconserved in the lectin fold(28);identicalresidues and conservativechangesatthesepositionsareboxed.Elsewhere, boxes representpositionsatwhich eitherat least five ofeightor at leastfourof six residuesareidentical.

To confirm the genomic structures of the recombinant

viruses,DNAextracted fromWR,vSAD7,andvSAD9virus cores was digested with KpnI, and the resolved fragments were transferred to nitrocellulose. Blots were probed with

32P-labeledDNA representing the TK gene,Ecogpt, or the sequence deleted from the endogenous SalL4R gene in

pSAD8 (Fig. 4). The latter probe detects the endogenous

18-kbKpnIfragment inWRand vSAD7 butnotvSAD9(Fig. 4A,lanes 1 to3),thusverifyingthat SalL4R ispresentat its natural locus in WR and vSAD7 but has been deleted in

vSAD9. vSAD7 and vSAD9 DNA contain an additional

fragment ofapproximately 14 kb as a result of the ectopic insertion of SalL4R within theTKlocus. This is confirmed by probingwith TK DNA(lanes4 to6).Thewild-type 16-kb

KpnI fragment containing TK is replaced in vSAD7 and

vSAD9 with fragments of approximately 14 and 4 kb as a result ofthe presenceofanadditional KpnI site within the

inserted DNA. The insertion of the Ecogpt gene into the endogenous SalL4R locus of vSAD9 also introducesanother

KpnI sitesothat two newfragmentsaregenerated. Onlythe 2.3-kbfragmentis detected with the Ecogpt probe (lane 9), andthisfragment is absent inthe otherviruses(lanes 7and 8). No other genomic rearrangements were detected by ethidiumbromide staining of KpnI-digested DNA(data not

shown).

IfSalL4R were required for growth of vaccinia virus in

tissue culture cells, plaque formation by vSAD9 should be

IPTGdependent.To testthis, confluentB-SC-1monolayers were infected withWR, vSAD7, or vSAD9 and maintained

for2days inthepresence orabsence of5 mMIPTG(Fig.5). WRand

vSAD7

formednormalplaques withorwithoutthe

addition of IPTG. However, normal plaque formation by

vSAD9,whichcontainsonly the inducer-dependentSalL4R gene, wasdependenton the presence ofIPTG. If IPTG was

omitted, only tiny plaques were produced. vSAD9 was

dependentonIPTG for normalplaque formation in allcells

tested(TK-143, RK-13, CV-1,and WI-38).

SalL4R

isnotrequired for infectious INV production but is necessary forvirus dissemination. To examine the cause of

the tiny-plaque phenotype of vSAD9 in the absence of

SalL4R geneexpression, the production of infectious virus

A 1 2 3 4 A C G T

[image:5.612.149.465.79.239.2]

-ACATTAATAAATOAAATCGCTTAATAGACAAACTGTAAGTAGGTTTAAG M K S L[N RO0T VS R F K

FIG. 3. (A) Si nuclease protection analysis of Sa1L4R

tran-scripts. A5'-radiolabeled probe (lane 1) (preparedas described in Materials andMethods)washybridizedwithyeasttRNA(lane 2)or

vaccinia virusearly(lane 3)orlate(lane 4)RNAand thendigested

with Si nuclease. Nuclease-resistant fragments were

electro-phoresed alongside anM13sequencingladder(lanes A, C, G,and T),and anautoradiographis shown. (B) Probepositionrelativeto the SalL4R ORF (M), the nucleotide and deduced amino acid sequences at the 5' end of the ORF, the site of transcriptional

initiation(****), and thedirection oftranscription(re-).

Consensus

CD23 El

AFPACP

ACB

vV

FPV Bam2 FPV Bam1

FPV Bam8

WIG¢SACLQ1A

$SEuT

I INQNRKIP NIIVDE

EN

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A 1 2 3 4 5 6 7 8 9

12

am

4 _ue 3 -2 _

m a,

4-

.~

.-we

..

B

o.0K I r 1KK

K; K; K K

TK

Ii-I'

l;--ir-K K

%SAJ1)7 "I _

iutdSal1S4R

EII_

S.al1.4R

K

--i

K K :-, K

1, "I

,-43 K---3 9K 23K K K KK K K

zdS11.4R F.G,([IT'

FIG. 4. (A)Southern blot of virus genomic DNA. DNAisolated from virus cores was digested with KpnI, and fragments were

resolvedon a0.8%agarosegel beforebeing transferredto nitrocel-lulose. DNAwasfrom WR (lanes 1, 4,and 7), vSAD7 (lanes 2, 5, and 8),orvSAD9 (lanes3, 6, and9). Filters wereprobedwith the

sequencedeleted from the endogenous SalL4Rgene(lanes 1 to3) withDNAfromthe TKgene(lanes 4to6),orwith the Ecogptgene

(lanes7to9). Sizes of the32P-radiolabeledladderoneach blotare

shown inkilobases. (B) Structures and sizesoftherelevant KpnI fragments containing the TK gene (El), Ecogpt gene (111111),

SalL4Rgene(Sal L4R; 1),andIPTG-inducible SalL4Rgene(ind

SalL4R; M) in WR, vSAD7, and vSAD9.

was followed during a single-step growth curve in the

presence or absenceofIPTG. Virus vSAD7 was usedas a

control since it also contains the IPTG-inducible 4b

pro-moter,asecondcopyofSalL4R,and thelacIrepressor gene

withintheTKlocus.B-SC-1 cellsinfectedat10 PFUpercell

with vSAD7 orvSAD9withorwithout IPTGproduced the

same amount of INV (Fig. 6A and B). SalL4R is not, therefore, essential for the synthesisof infectious virus.

Inaddition torequiringtheproductionof infectious virus particles, plaqueformationrequirestheir release andspread tosurrounding cells. Since SalL4Ris notrequiredfor INV synthesis but is clearly necessary for plaque formation, it seemed likelythat this protein plays arole in virus dissem-ination. To examine thispossibility, the kinetics of vSAD7 and vSAD9 replication during multiple cycles of infection

wasmonitored in thepresence orabsence of IPTG(Fig. 6C andD). IPTG hadnoeffectonintracellular virusproduction

overmultipleinfectiouscycleswhenanendogenousSalL4R

gene was present (vSAD7; Fig. 6C). With vSAD9,

replica-tionduringthe first 12hpiwasalso IPTG independent (Fig.

6D). However, by 24hpi, vSAD9 exhibited an 82% reduc-tion in theamountof infectious virus recovered from within

cells relative toinfectionsperformedin the presence of IPTG

(Fig. 6D). This decrease in virusrecoveredduringsecondary stages of multiple-step growth supports a role for SalL4R in virus dissemination in culture.

The effect ofSalL4R expression on EEV formation was

investigated by measurementof the virus present in super-natant of infected cells during low-multiplicity-of-infection

growthcurves. Intheabsence of SalL4R expression, there was an 87% reduction in supernatant virus at 36 hpi

com-paredwiththelevel in the presence of SaIL4Rexpression. Despite this,theproportion of supernatant virusoutof total virus did not fall without SalL4R expression, since the formation of INV was also restricted as a result of dimin-ished virus spread.Because the WR strain of virus produces very little EEV, e.g., 0.3% in RK-13 cells (32), it was

surprisingto find 5% oftotal progenyvirus inthe

superna-tant at the end ofthe growth curve. Ourinterpretation of

these data is thatsupernatant virus represents both EEV and some INV released by cell lysis which masks EEV

forma-tion. EEVformationwasthereforeanalyzed bythe alterna-tiveapproach of electron microscopy withininfected cells.

Intracellular wrapping of INV with Golgi-derived mem-brane requiresSa1L4R. The diminished virus spread in the absence of SalL4R could be due either to a block in the

production or release of EEV or to reduced infectivity of

released EEV. To distinguish between these possibilities,

EEV formation during a single infectious cycle of vSAD9

with or without IPTG was directly visualized by electron

microscopy. Representativeelectron micrographswhich il-lustrate the intracellularmorphogenesis ofvSAD9 are shown

(Fig. 7). By24hpi inthepresenceofinducer,all previously reported stages of vaccinia virus morphogenesis (20, 26)

wereobserved invSAD9-infectedcells(Fig.7D toF). In the

absence ofIPTG, all earlystages of vaccinia virus morpho-genesis,

including

formation of virus factories, lipid cres-cents, immature

particles,

immature

particles

with

nucle-oids,

andmature

INV,

werefound

(Fig.

7 Ato

C),

consistent with the growth curve data. However, without SaIL4R expression, no virions double wrapped with Golgi-derived membranewerefounddespite extensive searches. This form of viruswasabundantincrosssections ofcellularmicrovilli

in cultures infected in the presence of IPTG(Fig.7E andF). Similarly, only in the presence of IPTG could

double-wrapped virions be seenfusing withthe plasma membrane and

being

released as mature EEVfrom the surface ofthe

infected cell (Fig. 7F). These observations confirm that

SalL4R

plays

an

integral

role in the

wrapping

and/or associ-ation ofINV with

Golgi-derived

membranes and is neces-saryforthe

production

ofEEV.

SalL4Rencodes late22Kto24Kglycoproteins. To charac-terize the SalL4R gene product(s), a

polyclonal

antiserum was raisedagainst a

TrpE-SalL4R

fusion proteinand used forimmunoblottingofextractsfrom WR-orvSAD9-infected cells

(Fig.

8).Inextractsfrom cells infected withWRorwith

vSAD9in thepresence of IPTG(lane 1 or3,

respectively),

theanti-SalL4R serum identified a

triplet

of bands of22K, 23K, and 24K which were not found in extracts of cells infected with vSAD9 without IPTG (lane 5) or in

mock-infectedcells(lanes7 and8).BecauseSalL4Rcontainsasite

for N-linked

carbohydrate,

the

possible

glycosylation

of the

SalL4R

protein

was

investigated.

In the presence of

tuni-camycin,

the three bands

(22K

to

24K)

were

replaced by

a

singlebandof19K,the

predicted

sizeoftheSalL4R

primary

translation

product.

These

analyses

revealed that the

SalL4Rgene encodesa

glycoprotein

present in three forms

of

22K, 23K,

and 24K.

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Inducible SalL4R +

Endogenous SalL4R

vSAD9

+

vSAD7

oe].1 .s~

GP$,e +IPTG

-IPTG

WR

t,W 4e*4 / *

*

(hii *K

; ^ w I ?t '

t w#

*: f

: -,. f : R

PX. (A"'''.

*;...-:

..as,

...

FIG. 5. Plaques formed by WR, vSAD7, or vSAD9 after 48 h onB-SC-i cells in the presence (+IPTG) or absence (-IPTG) of5 mMIPTG. Cell monolayers were stained with 0.1% crystal violet in 15% ethanol. The presence (+) or absence (-) ofan inducible SalL4R geneor endogenous SalL4R gene within each virus genome is indicated.

Theprocessing and stability ofthe SalL4R protein were

examinedby pulsing infected cells with [35S]methionine for 15 min, chasing for periodsup to3h, and

immunoprecipita-tion (Fig.

9A). A

protein

of 23K was immunoprecipitated withanti-SalL4R antibodyfrom cellsinfected withvSAD9 in

A

loA

I106#

_{

_\

f

-0.-- vSAD7+IPTG

4 vSAD7-IPTG

the presence of IPTG(lane 2) butnot if IPTGwas omitted (lane 3), nor was it precipitated from uninfected cells (lane

1). Pulsing ofWR-infected cells for 15 minshowedthatthe

protein

wasalready inaglycosylated form (lane 4) andmust

therefore be rapidly processed. This formwasfairly stable,

B

la

0

-0-- vSAD9+ITG

- vSAD9O IPTG

0 10 20 30 40

hpl

C

0.

10

D

0.

-0-- vSAD7+IPTG

--- vSAD7-

IPTG

0 10 20 30 40 50 0 10 20 30 40 50

hpl hpi

FIG. 6. Growth curves of vSAD7 and vSAD9 in the presence and absence of 5 mM IPTG.B-SC-1monolayers were infected with vSAD7 (A)orvSAD9 (B) at 10 PFU per cell in the presence or absence of 5 mM IPTG, and the production of infectious virus particles was monitored duringasingle infectious cycle (Materials and Methods). (C and D) Replication of vSAD7 (C) or vSAD9 (D) inB-SC-1 cellsafter infection at0.001 PFU per cell and incubation in the presence or absence of 5 mM IPTG.

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W:

+.o,$WsW$

- Sic,#t_ _4e'i * .i'

0t _ w_

*''4vs

';fo.

_

s^%-'-/i

; ;; s

; v v liit W« vF

.. < *. _,i

.s s s jei t_ .

SS

4|f:w

*'-%Xf

_ ^ ot

t

.F _ <,^ w.

r

.0'A>..g't,...;

.J-.* . '' _

_.._ _.

.) Q "' t- _

.t0ti.-

>,

1_-w v Si__.

FIG. 7. Morphogenesis of vSAD9. B-SC-1 cells infected at 25 PFU per cell with vSAD9 with or without 5 mM IPTG were incubated for 6,12, and 24 h before being fixed with 2% glutaraldehyde, embedded, sectioned, and observed by electron microscopy. (A) Section of a cell 24hpi in the absence ofIPTGshowing virus factories and an INV particle (4). Numbers1to3are asfor panel B.Magnification, x5,278. (B) Detail ofavSAD9 virus factory without IPTG at 24 hpi. 1, Partially formed lipid crescents; 2, early immature particle completely surrounded by lipid; 3, immature particle containing condensed nucleoid. Magnification, x32,760.(C)Mature INV (indicated by arrow) produced in the absence ofIPTG, 24hpi. Magnification, x32,760. (D) Mature INV produced in the presence of IPTG, 24 hpi. Magnification, x32,760. (E) Cross section ofamicrovillus ofavSAD9-infected cell in the presence of IPTG at 24 hpi showing multiple INV surrounded by double membranes (indicated by arrow). Magnification, 32,760. (F) Cell at 24 hpi in the presence of IPTG. 1, INV wrapped in two layers of Golgi-derivedmembrane; 2, egress of EEV after the outer membrane surrounding a mature virus particle has fused with the plasma membrane of the cell. Magnification, x65,520.

since its decay during the chase (lanes 5 to 7) broadly reflected thedecrease intotal labeled cellprotein (Fig. 9B). Inthepresenceoftunicamycin, the 23K proteinwasabsent

and replaced with the 19K precursor, confirming that the

singlepotential site for N-linked carbohydrate is utilized. In addition, thereweretwolarger forms ofapproximately 40K thatwerenotseenby Western blotting. Apossible

explana-tion is that these are not the SalL4Rprotein but represent proteins which complex with SalL4R if normal processing is

blockedby tunicamycin. They arecoprecipitated with anti-SalL4R antibody, but the complex can be dissociated by SDSand/or

reducing

agents. These

proteins

are

presumably

virusencoded, sincetheyareefficientlylabeledatlatetimes during infection when host protein

synthesis

in inhibited.

Thetotallabeled cell extractsused forimmunoprecipitation (Fig. 9B) also showed nochanges in the

profile

ofvaccinia

viruslateproteinsin the absenceofSalL4R(lanes2and3). Thisfinding is consistent with the formation of normal levels

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IPTG

Tunicamycin - + - + - + -

-1 2 3 4 5 6 7 8

24kD 23 kD 22kD -19kD

-FIG. 8. Immunoblot analysis oftheSalL4R glycoproteins. Cells

wereinfectedfor 24 h with WR (lanes 1 and 2)orvSAD9 (lanes 3to

6) or were mock infected (lanes 7 and 8), in the presence (+) or

absence (-) of5 mM IPTG and/or 1 pLg oftunicamycin perml, as

indicated. Extractswereresolved by SDS-polyacrylamide gel

elec-trophoresis, transferred tonitrocellulose, and incubated with

anti-SalL4R serum, and immune complexes were detected by using

alkaline phosphatase-conjugated donkey anti-rabbit Ig (Materials and Methods). The molecular sizes of the observed proteins are

shownin kilodaltons.

of INV and with the electron microscopy data showing that this proteinfunctions late in viral morphogenesis.

TheSalL4R protein is associated with EEV particles. The SalL4R gene products have characteristics similar to those

A

465i

40 kD

30

,,

23 kD

-19kD

14 *

L 1 2 3 4 5 6 7 8

B

-_

FIG. 9. Pulse-chase analysis of the SalL4R protein. Uninfected

cells(lane1)orcellsinfectedwith vSAD9in thepresence(lane 2)or

absence(lane3)of5 mM IPTG,orwithWRinthepresenceof1,ug

oftunicamycinperml(lane8)orwithout drugs (lanes 5to7),were labeledwith [35S]methioninefor 15 minat6hpi. Cells wereeither harvesteddirectly(lane4)orchasedwith2 mMmethioninefor1, 2,

and 3 h before harvesting (lanes 5 to 7, respectively). Extracts

(preparedasdescribedin MaterialsandMethods)were immunopre-cipitated with anti-SalL4Rantiserum (A) andrun in parallel with aliquotsoftotalextracts(B)on a15%SDS-polyacrylamidegel. The

positionsof migration of14C-labeled protein standards areshown (laneL;indicatedin kilodaltons).

of a group of glycoproteins (20K to 23K) that are constitu-ents of the envelope of EEV (31). This similarity and the

demonstration that the

SalL4R

gene is required for EEV

formation (this report) suggested that the SalL4R

proteins

might be components of EEV. Thispossibilitywasanalyzed by immunogold labeling of purified virus particles (Fig. 10). EEV particles incubated with

anti-SalL4R

Ig became liber-ally decorated with 10-nm gold conjugated to goatanti-rabbit Ig (Fig. 10A and C) but not if unrelated rabbit Ig was

substituted for anti-SaIL4R Ig (Fig.

10F).

Higher magnifica-tion showed that SalL4R is closely associated with the membranous material surrounding and protruding from the virus particle (Fig.

10C).

To increase the antibody speci-ficity, the

anti-SalL4R

Ig fraction was affinity purified (Ma-terials and Methods) and shown to still efficiently label EEV

(Fig.

10B).

In contrast to the labeling of EEV, the vast majority of purified INV particles, which have a regular

brick-shaped morphologycompared with the more pleomor-phic EEV, remained unlabeled (Fig.

10D).

Very occasionally (<1%), a single labeled virus particle could be observed among several unlabeled virions (data not shown). This is

likely to represent a double-membrane wrappedvirion from the cytoplasm of infected cells which copurified with the INV. To allow antibody access toepitopes protected by the virus envelope, the virions were washed in 50% ethanol prior to incubation with

Ig.

If ethanol was omitted from the protocol, the specific

anti-SalL4R

Ig still recognized EEV (Fig.

10E)

but not INV (data not shown), confirming that the protein is present on the envelope of the EEV particle.

DISCUSSION

A vaccinia virus gene predicted to encode a type II transmembrane glycoprotein with homology to C-type ani-mal lectins (48) has been characterized. Si nuclease protec-tion analysis showed that the gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. An antibody to the SalL4R ORF raised against a bacterial TrpEfusion protein identified three proteins, 22K, 23K, and 24K, in infected cells. In the presence of

tunicamy-cin,

these proteins were replaced by a 19K protein, demon-strating that the 22K to 24K species are glycoproteins containing N-linked carbohydrate. The nature of the proc-essing of the primary translation product into the three forms remains unclear but might involve different types of proc-essing of core carbohydrate in the Golgi (38) or include other types of protein modification. Certainly, core glycosylation occurs rapidly, since the 19K form was not seen after a

15-min

pulse. Three polypeptides are seen by immunoblot analyses, but only one is seen by immunoprecipitation of cell extracts (Fig. 9A), possibly because of masking of epitopes in some of the glycosylated forms. Of interest was the coprecipitation of two other proteins of roughly 40K with SalL4R after pulse-labeling in the presence of tunicamycin. The failure to detect these proteins by Western blotting showed that they are not a complex containing SalL4R, and their efficient labeling late during infection indicated that they are virus encoded. Perhaps they are other virus glyco-protein precursors which aggregate in the endoplasmic

re-ticulum as a result of incorrect folding in the presence of tunicamycin.

The SalL4R glycoproteins were shown by immunogold labeling to be present in purified EEV of IHD-J but not INV (Fig. 10). Since the anti-SalL4R immunoglobulin reacted with EEV particles with nonpermeabilized membranes and with membranous

extensions,

the SalL4R glycoproteins are J. VIROL.

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A

C

o.

J v

0

41.

.1

''A..." 1,

-.Z'

1 t

0

.i ,:A

a0 0

D

E

*0

F

FIG. 10. Immunogold labeling of purified virus particles. Purified virus was incubated with either wholeanti-SalL4Rantibody (A, C and D), affinity-purified anti-SalL4R (B and E), or unrelated rabbit Ig (F). Bound antibody was detected with 10-nm-gold-conjugated goat anti-rabbit Ig. All panels show EEV except panel D, which shows INV. In panel E, the virion membranes were not permeabilized with ethanol prior to incubation with antibody. Magnifications are x65,520 (A, B, D, and E), x32,760 (F), and x127,400 (C).

present in the outer envelope of EEV and very likely represent the group of 20K to 23K glycoproteins shown

previously to be components of this envelope (30, 31).

SalL4Ris the second vaccinia virus gene shownto encode glycoproteincomponentsofEEV,the otherbeing the virus hemagglutinin (36, 46).

Toaddress the essentiality and function ofSalL4R in the

virus life cycle, we attempted unsuccessfully to isolate a

virus nullmutantinwhichtheSalL4Rgene wasreplaced by

the Ecogpt gene linked to a vaccinia virus promoter. This

method has beenused todeterminethe in vitroessentiality of other vaccinia virus genes (1, 21, 47), and the result

suggested that the SalL4R gene was required for virus replication.Todirectlytestthispossibility,avirus(vSAD9)

was constructed which conditionally expressed the SalL4R ORFfromanIPTG-inducible vaccinia virus 4bpromoter(41)

and from which the endogenous copy of the ORF was

deleted. Ifthegene were

essential,

thevirus would

replicate

only

in the presentof IPTG and the stageatwhich

replica-tion is arrested without IPTG could be studied. This

ap-proachhas been used toanalyzethefunctions ofthe 14Kand

l1K proteins in vaccinia virus

replication

(42,

58).

The

formation of normal

plaques

by

vSAD9 was shown to be

dependent on

IPTG,

with

only

tiny plaques

formed if the SalL4R gene was not

expressed.

Nevertheless,

the

single-step growth kinetics of this virus with or without IPTG

showed that normal amounts of INV were

produced

and

thereforetheSalL4Rwasnonessential for the

production

of

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infectious INV. This result demonstrates the

danger

in

assigning

gene

essentiality

on the basis of the inability to

isolateanullmutant.

By

this

criteria,

gene

encoding

proteins involved in virus dissemination

(14K, 37K,

and

SalL4R),

as

opposed

tothe

synthesis

ofinfectious virus

particles,

would be

incorrectly

classified as

being

essential for virus replica-tion.

Thefailureto

produce

normal

plaques despite synthesis

of

wild-type

levelsofINVhas been

reported

with virusgrown in the presence ofIMCBH

(44)

and with avirus inducibly

expressing

the 14K

protein (42).

Inbothof these situations, the

production

of EEV was

severely depressed, implicating

EEVasnecessaryfor virus

plaque

formation.Furthermore,

comet

formation,

ameasurementof efficient virus spread in

vitro,

isaproperty of virus strains

yielding higher

levels of

EEV

(32).

Theseobservations

suggested

that the failure of

vSAD9toform normal

plaques

in the absence of IPTGmight be duetothe lackofEEV

formation,

and thereductions in supernatantvirus andINV

during

the laterstagesof

multiple

step

growth

curves are consistent with this conclusion. Electron

microscopy

confirmed that in the absence of SalL4R

expression,

INV

particles

werenot

enveloped by

a

double

layer

of

Golgi-derived

membrane late

during

virus

morphogenesis.

These

double-enveloped

intermediate

medi-ates virion egress

by

fusing

the outermost membrane

layer

with the host cell

plasma membrane, allowing

releaseof the

EEVform of the virus

(20).

The

requirement

fortheSalL4R

glycoprotein

inINV

envelopment

could

explain

why

glyco-sylation

inhibitorspreventthe release of vaccinia virusEEV

(35).

EEV is

biologically significant

during

thedissemination of

poxvirus infections,

since antiserumraised

against

livevirus or

purified

EEV

envelope

protects

against

lethal doses of

virus,

while antiserum raised

against

inactivated INV does

not

(2, 3,

32).

Additionally,

the EEV

envelope

is

important

fortheenhanced

spread

of virus

by

mediating

efficient fusion with uninfected cells

(8).

If the SalL4R-encoded

proteins

function as

lectins,

they

might

contribute to the broad cell

tropism

of vaccinia virus in away similartothat

by

which

the influenza virus

hemagglutinin

(29)

or

herpes

simplex

virus

glycoprotein

C (17) contributesto thebinding of these virusestosialic acid-or

heparin sulfate-bearing

cells,

respec-tively.

The

availability

of the SalL4R conditional lethal

mutantshould notallow the

study

of the role ofEEV in the

spread

ofavaccinia virus infection in vivo.

SalL4R is the third

protein implicated

in the

wrapping

of

INV with

Golgi-derived

membrane. A mutation inthe

acy-lated 37K

major envelope protein

was found to confer resistancetothe

drug IMCBH,

whichprevents wrappingand release

(44).

Similarly,

thevaccinia virus14Kprotein

previ-ously

foundto bethetargetof

neutralizing

antibodiesand to

be associated with the INV membrane is also

required

for

EEV

envelopment

and egress (39, 42). Whether any inter-action exists between these three

proteins

is unknown. A

hydropathy

study

of 37K

predicts

thatapotential

cytoplas-mic domain of

approximately

130 amino acids could be

available for

binding

to an INV-associated protein (19),

while SalL4R ispredicted tohave a 11-residue cytoplasmic

domain

(Fig. 1).

The

fusogenic

properties of14K, which is

knownto form trimers (40), may indicate that this protein caninteract withcomponentsof theenvelopeand during the

wrapping

process and confer stability during virus release.

The

production

ofSalL4R and 14Kinducer-dependent mu-tants now facilitates an

investigation

into any interaction between 14K and either

SalL4R,

37K, or SalL4R-37K

complex.

Vaccinia virus hasmultiple mechanisms

allowing

success-ful infection of the

host,

ranging

from inhibition of host immune response to genes enabling a wide host range and stimulation of macromolecular

synthesis (6).

Deletion ofa

number of thesegenes whicharenonessential for

growth

of

the virus in tissue culture has resulted in

attenuated,

less virulentforms of the virus (5, 7). However, such viruses still

have the capacity to disseminate

throughout

the infected host. The SalL4Rmutant virus(or similar viruses grownin

cell lines providingthe complementing SalL4R

protein)

has potential as a rationally

designed

safe

poxvirus

vaccine

vector, since it has a severely restricted abilityto spreadin vitroandis predictedtohaveasimilarphenotype in vivo.In some respects, this application is similar to the use of avipoxvirusrecombinants inmammalian cells inwhich

only

abortiveinfectionsareestablished

(51).

Suchavectorwould

still allow the expression of recombinant

antigens

within

infected cells andsois

likely

topossessthe

immunogenicity

ofalive virus butwouldbe lessableto

spread

to

secondary

sites of infection and to cause accidental transmission to contacts of vaccinees.

ACKNOWLEDGMENTS

WethankD. Fergusonand L.Cohen-Gould for help with electron microscopy, L. Van Houten and H. Edwards forphotography, J.F. Rodriguez and P. Traktmanfor helpful advice, and P. Traktman and K. Lawfor criticalreading of themanuscript.

S.A.D. was the recipient of an MRC AIDS DirectedProgramme Research Studentship, and G.L.S. is a Lister Institute-Jenner Re-searchFellow.

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