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Viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus.

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JOURNAL OFVIROLOGY,

JUlY

1993,p. 4415-4420 0022-538X/93/074415-06$02.00/0

Copyright © 1993,American SocietyforMicrobiology

Viral

Liposomes Released

from

Insect Cells

Infected with

Recombinant Baculovirus Expressing the Matrix

Protein of

Vesicular

Stomatitis Virus

YAN

LI,"

LIZHONG LUO,1 MANFRED SCHUBERT,2 ROBERT R. WAGNER,3 ANDC. YONG KANGl*

Department ofZoology, Faculty of Science, and Department ofMicrobiology and Immunology, Facultyof Medicine, The University ofWestern Ontario, London, Ontario, CanadaN6M 5B7';National Institute of

Neurological Disorders andStroke, Bethesda, Maryland 208922; andDepartment of Microbiology andCancer Center, University of

Virginia

SchoolofMedicine, Charlottesville, Virginia 22908'

Received30 November1992/Accepted 16 April 1993

The matrix (M) proteinofvesicularstomatitis virus(VSV)hasbeenfoundto promoteassembly and budding of virionsaswellasdown-regulatingof VSVtranscription. Large quantitiesof Mproteincan beproduced in

insectcellsinfectedwith recombinantbaculovirus expressingtheVSVMgeneunder controlofthepolybhedrin

promoter. Analysis by pulse-chase experiments and density gradient centrifugation revealed that the [35SJmethionine-labeled M protein synthesized in insect cells is released into the extracellular medium in association with lipid vesicles (liposomes). Electron microscopy and immunogold labeling showed that M protein expressedininsect cellsinducedthe formationonplasma membraneof vesiclescontainingMprotein,

whicharereleased fromthe cellsurfaceinthe form ofliposomes.Thebaculovirusvectoritselforrecombinants expressing VSVglycoprotein (G) or nucleocapsid (N) protein did not produce the formation of vesicles in infected cells. Thebaculovirus-expressedMproteinretainsbiologicalactivityasdemonstrated by itscapacity

toinhibittranscriptionwhen reconstituted with VSVnucleocapsidsinvitro.Thesedatasuggestthat M protein has thecapacitytoassociatewith theplasmamembrane ofinfectedcellsand, insodoing,causesevagination

of the membranetoformavesicle which isreleased from the cell. This observation leadstothepostulate, which requiresfurtherproof,that the VSV Mproteincaninduce the formation andbuddingofliposomes fromthe

cell membrane surface.

The prototype rhabdovirus, vesicular stomatitis virus (VSV), contains a membrane-enclosed ribonucleoprotein

(RNP) core with a negative-sense unsegmented RNA

ge-nome that codes for five structuralproteins(9, 10, 23). Two of the fivevirallycodedproteins aremembraneassociated: (i)theintegral, externallyorientedglycoprotein (G) spikeis theligandthatrecognizeshost-cellreceptor and is themajor type-specific antigen which gives rise to and reacts with neutralizing antibody(12); and(ii) theperipheralmatrix(M) protein lines the innersurface of the virionlipid bilayer in close association with the RNP core (22). The three other VSV proteins, comprising the RNP core, are the major nucleocapsid (N) proteinand theminorlarge (L) proteinand phosphoprotein (P, formerly designated NS) (10, 23),which collectivelyserve as theRNA-dependent RNApolymerase activity (7). Alsotightlyassociatedbyionicbonds with the RNPcoreis the Mprotein,whichservestowindupthe RNP

coreintoatightcoil(19). The association ofMproteinwith RNPcoresalso results indown-regulationoftranscriptionof genomicRNA(3).

Anotherpresumedpivotal function of theMproteinisin assemblyof the RNP-Mcomplexwith the host cellplasma membrane containing the newlyinserted G protein, which

canalso be releasedfrom the cell surfaceby proteolysis (4). Thepositively chargedMproteinhasbeen foundtointeract with and bind to the head groups of negatively charged membranephospholipids, primarily phosphatidylserine (24). Still another recently described function of the VSV M proteinis its latecytopathiceffect, probablydueto

interac-* Correspondingauthor.

tion with cytoskeleton, resulting in typical rounding of ordinarily polygonalcells afterVSVinfection (2). Although ithaslong beenassumedthatVSV M protein istheprincipal factorinpromotingvirionmorphogenesis and budding from the surface membrane (23),no concrete evidence supports

this hypothesis. The experiments reported here were

de-signedtodetermine whether the Mproteinalonecaninduce

buddingoflipidvesicles from the cell surfacemembrane. Inordertoexpresslargeamountsof theVSVMproteinin SF9 insect cells, the entire M gene sequence was excised

fromapreviouslydescribedplasmid, pYL-OM79 (15), with

BamHI and KpnI, blunt ended with Klenow polymerase and phage T4 DNA polymerase, and inserted into the BamHI site of baculovirus transfer vector pAcYM1 (18) blunt ended with Klenow polymerase. The resulting plas-mid, pAcYM1-M, was cotransfected into SF9 cells with

wild-typeAutographa californicanuclearpolyhedrosis virus (AcNPV)viral DNA. Aplaque-purified cloneof the

recom-binant baculovirus vAcVSV-M was used to express M

protein in infected SF9 cells. The M protein expressedby vAcVSV-Mwas analyzedat 1, 2,and 3daysafterinfection of SF9 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue stainingandbyWesternblots(immunoblots)ofextractedor

partially purifiedMproteinelectroblottedontonitrocellulose sheets. AlsoexaminedbyWesternblotanalysiswas

partic-ulate Mproteinreleased into the culturemedium ofSF9 cells infected with vAcVSV-M.

Figure1shows the timecourseof Mprotein synthesisby

vAcVSV-M after SDS-PAGE of infected SF9 cell extracts determined by Coomassie blue staining (Fig. 1A) or by

immunoblotting with monoclonal antibody 2 (MAb2)

di-4415

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A

Cell

mark M 1 2 3 Wt C

B

Cell

M 1

2

3

Medium

1 2 3 Wt C

97 86

-42

--

-~~~~~...

31-

21-14

am --- -_' ;

FIG. 1. Electropherogramsofdaily synthesisof VSV Mprotein expressedin SF9 cellsby recombinant baculovirus vAcVSV-M,detected by Coomassie blue staining of cell lysates(A),Westernblot analysis of cell lysates (Cell [B]),and particles inthe culture medium (Medium [B]).SF9 cells wereinfected withvAcVSV-M recombinant or a wild-type baculovirus control (Wt) atmultiplicities ofinfection of 5 PFU per cell. Thewhole-celllysates collected at days 1, 2, and 3 p.i. (lanes 1, 2, and 3, respectively) were analyzed bySDS-12%PAGE anddetected by staining withCoomassie blue(A) (16)orelectroblotted onto a nitrocellulose sheet forimmunoblottingwith MAb2(directedto Mprotein epitope 1) and detected with "251-labeledstaphylococcal protein A (Cell [B])(15). The presence of M protein in the culture medium at days 1,2, and 3 p.i. of vAcVSV-M-infected SF9 cells was determined by SDS-PAGE andMAb2immunoblotting of the pelletafter centrifugation in the SW50.1 rotor at 30,000 rpm for 1 h (Medium[B]).Bio-Rad molecular size markers (mark) in kilodaltons are shown at theleft, and the VSV virion marker Mprotein (lanes M)isshownatthe right. The controls are mock-infected SF9 cells (C) and cells infected withwild-type baculovirus AcNPV (Wt).

rectedtoepitope 1 of theMprotein (Fig. 1B).As canbe seen inFig. 1A, vAcVSV-M recombinant baculovirus infection of SF9 cellsexpressed largeamountsofastainableproteinthat comigrated withvirion Mproteinat-34kDa; the amount of stainable M protein synthesized appearedto bemaximalat day 2 postinfection (p.i.) and diminished by day 3 p.i., at which time there was a marked decrease in the amount of SF9 cell protein probably due to cell death (Fig. 1A). No stainable bands corresponding to those of M protein in uninfectedorwild-type baculovirus-infected SF9 cells were observed afterSDS-PAGE(Fig. 1A).

The identity of the M protein expressed in vAcVSV-M-infected SF9 cells wasclearly confirmed by reactivity with epitope 1-specific MAb2 by Western blots (Fig. 1B). Once again,maximumsynthesisofMproteinwasobserved atday 2 p.i., diminishing by day 3 p.i. It should be noted that expressed recombinant M protein migrated by SDS-PAGE slightlymoreslowly than the marker virionMprotein(lanes M).Moreover, the Mprotein expressedatday2p.i.showed a split band, conceivably due to differences in degree of phosphorylation,asistruefor theP(formerly NS) protein in mammalian cells (13).

Ofparticularinterest wasthefindingthatrelatively large amounts ofMprotein werereleased into the medium from SF9 cellsinfected with vAcVSV-M (Fig. 1B).Itwasfound that pelleted material present in the medium surrounding vAcVSV-M-infected SF9 cells containedaprotein migrating identically withMprotein and strongly reactive by Western blot with epitope1-specific MAb2 (Fig. 1B, Medium). Once again, the largest amount of M protein released into the mediumoccurred at day 2 p.i. and diminished by day 3 p.i. These results indicate that M protein is extruded from SF9 cells infected with vAcVSV-M expressing the VSVM pro-tein.

Thequestion whether the sedimentable form of M protein released from SF9 cellsinfected with vAcVSV-M could be duetocelllysates dispersed into the culture medium arose.

Light microscopy of these infected cells made cytolysis

unlikelyastheprincipal explanation for release ofMprotein

because the cells remainedrelatively intact until 2daysp.i. whenonly20% of the cells had begun toundergo lysis. By thethirddayp.i.,thecytopathiceffectwasabout70% (data

not shown). To shed some light on the question whether sedimentable M proteinwasgradually released from intact cells infected with vAcVSV-M, a pulse-chase experiment was performed to estimate the rate at which M protein is released. To this end, SF9 cells infected with vAcVSV-M were pulse-labeled with [35S]methionine for 30 min at 16h p.i.; the cells were then thoroughly washed and incubatedin chasemedium for24h.Atintervals after thechase, medium and cells were harvested separately, the cellswere lysed,

and the cell-free medium and the cell lysateweresubjected toimmunoprecipitation by addingexcess amountsof MAb2 directed toepitope 1 of the VSV Mprotein.

Figure2 shows electropherograms of35S-labeled M pro-teinimmunoprecipitated from cell lysates (Fig. 2A)or cell-free culture medium (Fig. 2B) harvested from vAcVSV-M-infectedSF9 cellsatchaseperiodsof0.5to24 hafterpulsing

with[35S]methionine.Asnoted, intracellular radiolabeledM protein was present in large amounts at the 0.5-h chase

period after the 16-hpulse; longer chases showedagradual

decline in cell-associated 35S-M protein from 4 to 24 h

postchase. By comparison, a small but quite significant amountofMproteinwasfound in extracellular mediumby

30 min after chase; thereafter, the amount of M protein gradually accumulated to high levels in the extracellular mediumduringthe 24-h chaseperiod.

These resultspointto aprogressive release ofMprotein

from vAcVSV-M-infected SF9 cells. These data also appear torule outcell lysis as an important factor contributingto release of M protein into the medium surrounding the infectedSF9 cells.

Thequestion whether the sedimentable Mproteinreleased from vAcVSV-M-infected SF9 cells is in the form of an

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A

0.5 1

2 4

8

16 24

M-

_mqm

___

B

0.5 1 2 4 8 16 24 hr

_ _ _m m __

NOTES 4417

FIG. 2. Pulse-chase analysis of[35S]methionine-labeledM protein synthesized in SF9 cells (A) and released into thecell culture medium

(B). SF9cellswereinfected with recombinantvAcVSV-Matamultiplicity of infection of 5 PFUpercell. At 16 h p.i., cellswereplaced in

methionine-freeGrace medium for 30min,afterwhichtheywerepulse-labeledwith[35S]methionine(250 ,uCi/ml) for1h.The cellswerethen washed and chased by incubation in complete medium fordifferent intervals of time upto aperiod of 24 h. At the chase timeintervals

indicated forseparatecultures,mediumand cellswereharvestedseparately and cell lysateswerepreparedasdescribed by Lietal. (14).Each

celllysateand itscorrespondingmediumweresubjectedtoimmunoprecipitation by addingexcessMAb2 (directedtoMproteinepitope 1)

to precipitate 35S-labeled M protein. The immunoprecipitated 35S-labeled M protein was analyzed by electrophoresis on SDS-12% polyacrylamide gelsandautoradiography. The slabgelinpanelBwasexposed twiceaslongastheoneinpanel A.

insolubleMproteinaggregateorisassociated withhost cell components, one of which could be cellmembrane, arose.

One methodtodetermine whether the Mproteinisreleased from cells in association with membrane lipids is sucrose

density gradient centrifugation. To this end, M protein released into the culture medium 48 h after infection of SF9 cells with vAcVSV-M was collected by sedimentation at 81,000x gfor 1 h and thepelletwaslayeredover a20to60% discontinuous sucrose gradient and then centrifuged at

120,000 xgfor 4 h. Aftercentrifugation, three visiblebands werediscernibleatthe interfaces of20/30%sucrose,30/40%

sucrose,and40/50%sucrose(datanotshown).Each of these bandswascollected and subjectedtoelectrophoresis on an

SDS-12% polyacrylamide gel. The resulting SDS-PAGE proteinswere electroblotted onto a nitrocellulose sheet for

Western blot analysis usingMAb2 directed toepitope 1 of the VSV M protein. Autoradiographs were prepared after exposure to

PI-labeled

staphylococcal protein A.

Figure3 shows the Western blotanalysisof the Mprotein in each of the visible bands in a 20 to 60% discontinuous

sucrosegradient aftercentrifugation. Asnoted, mostof the Mproteinreleased fromvAcVSV-M-infected SF9 cellswas

bandedatthe30/40%sucroseinterface(Fig.3,lane4),buta

significant amount of M protein was found in association with baculovirus particles, banding at the 40/50% sucrose

interface (Fig. 3, lane 5) (16, 17). These data indicate that most of the M protein released from vAcVSV-M-infected cells has a low density consistent with association of the released M protein with membrane lipids in a particulate

structure. Totest the postulateof an Mprotein-membrane complex released from cells infected withvAcVSV-M, the material that banded at the 30/40% sucrose interface was

subjected to asecond roundofcentrifugation. Thepurified

material thatreappearedafterrecentrifugationatthe30/40%

sucrose interface was collected andpelleted by centrifuga-tionat81,000 xg,and thepelletwasexaminedby negative-stain electron microscopy. As shown in Fig. 4B, the low-density fraction of M protein released from vAcVSV-M-infected SF9 cells contains numerous structures closely resembling lipoproteinvesicles.

These datasuggest thatMprotein expressedin SF9 cells

is released into the medium in association with cell

mem-brane in theformofliposomes. Thisobservation is

compat-ible withthehypothesis that the VSV M protein inducesthe formation from host cell plasma membrane oflipoprotein vesicles that are progressively released into the culture

medium. Theamountof M protein releasedfrom

vAcVSV-1

2

3

4

5

M

-4

FIG. 3. Westernblotanalysis of M proteininmedium after release fromvAcVSV-M-infected SF9 cells afterfractionation by

centrifuga-tion. SF9 cellsinfectedwithvAcVSV-Matamultiplicity of infection of

5 PFU per cellwere incubated for48 h, and the cell-free culture

mediumwas pelleted bycentrifugation at 81,000 x g for 1 h. The pelletedmaterialwas resuspended incoldphosphate-buffered saline (PBS) (500,ul) containing0.1%Tween20, 10 pLgof aprotininperml,

and 1 mMphenylmethylsulfonyl fluoride and leftat4°C overnight. The samplewaslayeredon a20to60%discontinuoussucrosegradientand

centrifugedin theSW41rotor at120,000xgfor4hat4°Casdescribed

by Luoetal. (16, 17). Threevisible bandswerecollected from the 20/30%, 30/40%,and40/50%sucroseinterfaces, diluted with cold PBS,

andrepelletedat81,000xgfor 1 h andsubjectedtoelectrophoresison anSDS-12%polyacrylamide gel. Afterelectroblottingonto

nitrocellu-losesheets,the Mproteinineach fractionwasidentifiedby

immuno-blottingwith MAb2directedtoMprotein epitope1.Autoradiographs

werepreparedafteradding125I-labeledstaphylococcal proteinA.Lane

1, purifiedMproteinfrom VSVvirions;lane2,totalpelletedmaterial from vAcVSV-M-infected cell culturemedium;lane3, 20/30%sucrose

gradient interface;lane4, 30/40% sucrosegradientinterface;lane5, 40/50%sucrosegradientinterface.

VOL. 67, 1993

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preparation of released vesicles. (A) Cells infected with wild-type baculovirus AcNPV (48 h p.i.); note polyhedrin inclusions bearing progeny

inece 48 h prvoul wit reobnn bauloiue exrssn VS prtei G oN,rsetvl. For imuogl laeig th hiecin

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morpho-4418 NOTES J. VIROL.

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NOTES 4419

genesis of the Mprotein-associatedvesicles, we resorted to electronmicroscopy.By this means,weexaminedSF9 cells by thin-section transmission electron microscopy at inter-vals after infection with M protein-expressing vAcVSV-M compared with cells infected with control wild-type baculo-virus. As additional controls, SF9 cells were examined by electronmicroscopyafter infection with baculovirus recom-binantsexpressingproducts of the VSV G gene, YMlVSVG (1),ortheVSVNgene,whichwasconstructed by excising the entire coding region of the wild-type VSVNgene from

pGN2(6)withXhoI, blunted with Klenowpolymerase, and

recloning into the blunt-ended BamHI site of pAcYM1. Othercellswereexaminedbythin-section electron micros-copy after coinfection with all three baculovirus recombi-nantsindividually expressing the VSV G, N, and M genes. ToidentifyVSVprotein associated with these vesicles, the sectioned cells were exposed to monoclonal or polyclonal

antibodies directedto M, G, orNproteins and then conju-gatedwith goat anti-mouse immunoglobulinG labeledwith

colloidal gold.

Figure4shows the electronmicrographsofsectionedSF9

cellsinfectedwithwild-typeor recombinantbaculoviruses, aswellasnegativelystained vesiclespreviouslydescribedas having been released into the medium from vAcVSV-M-infected SF9 cells (Fig. 4B). The formation of ringlike

vesicles emanating from the surface membrane is clearly shown inFig. 4C, depictingSF9 cells infected with

M-pro-tein-expressingvAcVSV-M. In contrast, novesicle forma-tion is evident on the surface of SF9 cells infected with

wild-typeAcNPV(Fig.4A)or onthat ofSF9 cells infected with baculovirusesexpressing VSV GorNproteins (Fig. 4E andF, respectively). These findings appearto indicate that

onlythe Mproteincausestheproductionofvesicles,which are then released from vAcVSV-M-infected cells into the

surrounding medium(Fig. 4B).

Figure 4D demonstrates that these vesicles induced in

vAcVSV-M-infected SF9 cells contain the expressed M

protein,asevidencedby labelingof the surface vesicles with MAb2(directed to epitope 1 of Mprotein) conjugated with goat anti-mouse immunoglobulin G labeled with colloidal

gold. Theshapeand size of theringlikevesicles associated with Mproteinarequite irregularbut average about 130nm in diameter; the immunogold labeling pattern is also quite thick, as evidencedby thedensityof thering, suggesting a multimericlayeringof theMproteinwithinpossiblystacked membranelayers(Fig. 4D).Examination of manysectioned cells failedto show anyvesicles associated with Mprotein

within the cytoplasmofvAcVSV-M-infected cells(datanot shown). Nordidimmunogold labeling provide anyevidence forthe presence ofGorNproteinin vesicles formed in SF9 cells coinfected with recombinant baculoviruses expressing

M,G,and Nproteins (datanotshown). These data support thehypothesisthat thebaculovirus-expressed Mprotein, in the absence of any other VSV genome products, finds its wayto and becomes associated with cell membrane. These results also supportdatafromcross-linkingstudiesshowing

thatMproteininteracts with membranebilayers by

electro-statically attaching tonegatively charged phospholipid ves-icles(24, 25).

Itis well documented thatbindingofwild-typeMprotein toRNPcoresresults indown-regulation oftranscription (3, 21).It wasof interesttodetermine whether the recombinant Mprotein expressed bythe baculovirusvectorin insect cells retained thebiologicalpropertytoinhibit VSVtranscription.

If so,this would

provide large

amountsofM

protein

needed forsuchstudies,which has beenalimitation in the past. To

this end, the transcription-inhibition activity of M protein extracted fromVSV virions and purified by phosphocellu-losechromatography(21) was compared with that of recom-binantbaculovirus-expressedMprotein purified by

ultracen-trifugation on a discontinuous sucrose gradient. Each of these sources of M protein was tested for its capacity to inhibit transcription after reconstitution with VSV RNP cores. When increasing concentrationsof virion M protein orbaculovirus-expressedMproteinwerereconstituted with RNPcores(180 ,ug/ml), transcriptional activitywas

progres-sivelyreducedto alevelof 50to60%inhibitionatMprotein concentrations of 200 ,ug/ml (data not shown). Clearly,

purified M protein extracted from virions or expressed by thebaculovirus recombinant exhibited almost identical pro-files oftranscription-inhibition activity. These results dem-onstratethatthe recombinantbaculovirus-expressedM pro-tein retains thebiological activityof the virion-associated M

protein, atleastasfarasdown-regulation of transcription is concerned.

Inconclusion,theseexperimentsindicate that the VSV M

protein has the intrinsic property ofpromotingevagination of a circumscribed region of the host cell cytoplasmic membrane, leadingtoliberationofliposomes.This property is consistent with the recent finding that perhaps 10% of

vector-expressed VSV M protein attaches to cytoplasmic membrane and isnotreadily dissociable (5). Clearly, these functions of the Mprotein are intrinsic andindependentof other VSVproteins.The Mproteinalso bindstoRNPcores in vitro (11, 20) and in vivo (5), an event that precedes

migration

of the RNP-Mcomplextothesurface membrane where the progeny virion buds (23). Alogical extension of these findings is that the membrane

evagination-vesicle-forming

function of the M protein is also the M protein

property that causes budding of progeny virions from the surface membrane of VSV-infected cells. Liposomes with associated Mproteinarealsoreleased from mammalian cells transfected with plasmids expressing wild-type VSV M protein (8), indicating that this property is not limited to insect cells. The mechanismby which M protein promotes vesicle formation, and perhaps budding, remains to be

determined,but solution of thisproblem has wide ramifica-tions.

Thisresearchwassupported bygrantsfromtheMedicalResearch Council of Canada and by USPHS grant R37 AI-11112 from the National Institute ofAllergyandInfectious Diseases.

We thank F. Wei for electronmicroscopy.

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Figure

FIG. 1.byVSVbaculoviruscell.by[B]).inepitope1, 2, the Electropherograms of daily synthesis of VSV M protein expressed in SF9 cells by recombinant baculovirus vAcVSV-M, detected Coomassie blue staining of cell lysates (A), Western blot analysis of cell lysa
FIG. 2.washedcellpolyacrylamideindicatedtomethionine-free(B). precipitate Pulse-chase analysis of [35S]methionine-labeled M protein synthesized in SF9 cells (A) and released into the cell culture medium SF9 cells were infected with recombinant vAcVSV-M at
FIG..4. prvoul releasedEr with.. vesicles. infectedwitcr reobnn (A) Cellsoe~~~~~~~~~. bauloiue.''_,nFlT,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A exrssn wild-type

References

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