Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

0022-538X/88/031008-08$02.00/0

Copyright ©O 1988, American Society for Microbiology

Development of Avian Sarcoma and Leukosis Virus-Based

Vector-Packaging Cell Lines

ANDREW W. STOKER* ANDMINA J. BISSELL

Laboratory of Cell Biology, Division of Biology and Medicine, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

Received 16September 1987/Accepted 13 November 1987

We haveconstructed anavian leukosis virus derivative witha5' deletionextending from within the tRNA

primerbinding site toa Saclsite in the leader region. Our aimwas toremovecis-acting replicative and/or

encapsidationsequencesand tousethisderivative,RAV-1*-, todevelop vector-packaging cell lines. We show

that RAV-14-canbestably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, wehave demonstrated that QT6-derived lines expressing

RAV-1*-

canefficiently package four structurally different replication-defective v-srcexpression

vectorsinto infectious virus, withverylowor undetectablehelper virus release. TheseRAV-l*--expressing cell linescomprise the first prototype avian sarcomaand leukosis virus-based vector-packaging system. The construction ofour vectors has also shown usthatasequence present withingag, thought to facilitate virus

packaging, is notnecessaryfor efficient vectorexpression and high virus production. We show that quantitation and characterization ofreplication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presentinganalternative to internal selectable vector markers.

Retroviral vectors have proved to be highly efficient for introducing clonedgenes into culturedcells and organisms. Recently developed retroviral vector packaging cell lines provide infectious, replication-defective (rd)viruses without helper virus contamination, thus avoiding potential problems withviral interference and helpervirus-induced disease(22, 24, 41). The mostextensively used system, the murine i2 cell line, has facilitated the transduction of genes into hemopoetic, neuronal, and embryonal carcinoma cells for studies ofhemopoetic reconstitution and for chromosomal marking (7, 11, 14, 29, 32, 37, 40, 42). Such work has enhanced theunderstandingofdifferentiation, celllineages, and thebehavior ofretroviral vectorsin vivo.

Our studies have centred upon the interaction of Rous

sarcoma virus (RSV) and the oncogene v-src, with both hatched andembryonicchicken tissues. We have shown that RSV-inducedtumorigenesis is both inhibitedin ovo(9)and

dependent upon wounding in hatched birds. (10). These unforseencomplexitiesofRSV-induceddisease raisea

num-ber ofquestions that could be addressed appropriately by usingrdv-src expression vectors. Althoughthere has been recent interest in its use, a packaging system designed specifically for avian sarcoma and leukosis virus (ASLV)

vectors is not presently available. The feasibility of

con-structingsuchasystem'with avian leukosis virus has been in some doubt because of the poorly characterized avian

leu-kosis virus RNA encapsidation region and the possibility thatatleastpartof theregion lies 3' tothesplice donor site withingag(31). The leader (L) region, however, is believed

tocontainatleastapartof thecis-actingRNAencapsidation

sequences(4i), sincetwonaturally occurringRSVpackaging mutants,TK15(27)andSe21Qlb (34),bothcontaindeletions in L(fora reviewsee reference6). Arecent study by Katz etal. also describedaspecific sequencewithin the Lregion necessary for RNA encapsidation in RSV (17). We show here thatremoval ofarelated sequencewithin the Lregion ofacloned avianleukosis virushelperis sufficienttoabolish

*Correspondingauthor.

virus replication in cis. Using this crippled helper virus, designated RAV-1-, we have establishedprototype pack-agingcelllines for ASLV-based vectors.

With sensitive immunocytochemical techniques (36) we

show that transfected pRAV-1h- directs the expression of viralproteins but doesnot supportreplicatingvirus

produc-tion from either chicken embryo fibroblasts (CEF) or the

quailline QT6 (25). Stable, RAV-14--expressing QT6lines have remained helper virus free since their development. These same cells were shown to package three different ASLV-based rd vectors into infectious virions in transient

assays, without significant helper virus titers. Packaging cells stably releasing vector-derived virus provide greater than i05 viruses per ml, with very little or no helper virus

release. We have thusdemonstrated both the feasibility of designing ASLV-based packaging cell lines and the

con-struction ofspecific vectorsforusein these cells. MATERIALS ANDMETHODS

Cell culture.QT6isachemicallyderivedtumorcell line of

the Japanese quail, free ofexogenous ASLV (kindly

pro-videdbyP.Vogt, University of Southern California, School ofMedicine, Los Angeles) (25). CEF were prepared from day 10 white leghorn embryos (specific pathogen free) as

described previously (3). All cells were routinely grownin medium 199(GIBCO Laboratories) supplemented with 10%

tryptose phosphate broth, 4% newborn calf serum, 1% chickenserum, and 100 p.gofgentamicinperml.

Manipulationof cloned viral DNA. All recombinant DNA manipulations wereperformed by standardtechniques (21). Proviral DNA inplasmid formis referred to with prefix p,

whereas the viruses expressed havenoprefix.

pRAV-1*-

(Fig.1and2).The clonedhelpervirusgenome

pRAV-LTR was'kindly given by B. Vennstrom (Differenti-ationProgramme, EuropeanMolecularBiology Laboratory, Heidelberg, Federal Republic of Germany). This plasmid containsacloned, permuted Rous-associated virus(RAV-1)

inserted asa Sacl fragment 3' toan avian erythroblastosis 1008

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R as S

L~

L*

C

_LTR- 9a9

Bs Bs Bs

R K SasR R|S B

I

I

1

Lp

I

pol env

L--pRAV-LTR LTR

pRAV-LTR

K

(a) (b)

(a) Sacl digest

(b) Remove RAV-I DNA /

(c) Self ligate

I

Kpn digest

Ligate 3' viral DNA to modified pucI9

|K

(a) BstEII digest and

remove fragment I

(b) Add SacI linkers (c) Ligate to RAV-I

DNA

-Exchange fragment 2 for EcoRIb fragment of PrA RSV

K So R 9g R a

pRAVsrc

I

0,S&,K

IN 2 3

Bs B--s Bs

Ba R B Ba

Sa

pRAVKpn3'

K

(a) Partial BstEII digest

(b) Remove fragment 3

(c) Self ligate

R Bs,S aRs Bs B

.14 1-

//I-pRAV-5

IWJ

(a) Kpn digest

(b) Isolate 5' viral fragment

Q

/

R K RBaB

RRA1K Ss R B

.1

I

s^

l-Q

I

pRAV-1WI

-(a) KpnI digest

(b) Isolate 3' viral fragment

K

FIG. 1. Schematic representation of

pRAV-14-

construction. Boxed regionsrepresent proviral DNA, RAV-1 (O) andAEV10(U).Single linesrepresentbacterialplasmid DNA. DuplicatedRNAencapsidationsequences

(Oi)

areindicatedin pRAV-LTR. Relevant enzyme cleavage sitesareindicated: B,BamHI(sites within RAV-1 DNA are notshown);Bg,BgII;Bs, BstEII; K,KpnI;R, EcoRI;5,Sacl;Sa,Sall.

L

RAV-LTR

RAy-lW-U5 I

I

_.,,,,,,g

-Dr7.aa

BstEll SacI BamHI

r-pr76gsg f

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sd

FIG. 2. Diagram representing 5' regions of pRAV-LTR and pRAV-li- proviruses: AEV10 DNA (_), RAV-1 DNA (mi and =!). TheU5 region of theAEV10 LTR is shown, with thetRNA

PBS, the noncoding leader region (L), and the startof the pr76xag coding region. sd, Splice donor site. The 5' region deleted from pRAV-LTRtoformpRAV-14- is indicated.

virus (AEV10) long terminal repeat (LTR) in pSV2gpt (26, 39). Due to the permuted structure of this provirus, two similar deletions were introduced to remove putative 4, sequences.Thedeletionin the 5'AEV10 L regionwasmade first by cleaving pRAV-LTR with SacI to remove RAV-1 sequences. The resulting plasmid pLTR was digested with BstEII to removethe L-regionsequence; SacIlinkers were added, and the RAV-1 DNA was ligated back to create pRAV-5'iP, containing the correct 5' deletion. Deletion of 4 sequence 3' of the RAV-1 LTR was initiated by cleav-ing pRAV-LTR with KpnI and ligating the 3' viral DNA fragment to amodified pUC19 plasmid (lackingEcoRIand SacI restriction sites). This plasmid, pRAVKpn3', was di-gested partially with BstEII and self-ligated to create

pRAVKpn3'*-,

which now contained the 3' deletion

(ap-proximately 100basepairs

[bpl

largerthan the 5' deletion).

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pAll LTR

_r

H

9ag9 Apol W-arc LTR

6

a c

M0S

PASrci LTR 4 v-arc * LTR

pASrc1l

B/S

pASrcneol

U3 RU5

RESV _|_RAV1

EcoRI

1 Kb

L--SH IB/BgI

pASrcneo6

LR-ac

FIG. 3. Replication-defectivev-srcexpressionvectors.pAll isapol-B77RSV;pASrclandpASrcneolarederived fromRAV-1and PrA RSVDNAand havechimericLTRs(inset contains theLTRsofpASrclandpASrcneol,showingboundaries ofRSVand RAV-1sequences). Agag, Apol, and Aenvaretruncatedgenes. Symbols:positionof deletedHindlllfragmentinpAll(A) andsplicedonor(0)andacceptor

(0)sites. Unique cloning sites invectorspASrclandpASrcneolareindicated. B,BamHI;S,Sall;H,Hindlll;TK pro,thymidinekinase gene promoter; neo,neomycin resistancegene.

To construct pRAV-1I-, 5' and 3' proviral halves were ligated together as KpnI fragments from pRAV-5'4i and pRAVKpn3O'%-, respectively.

pAll (Fig. 3).pAll proviralDNAwaskindly givenbyD. Gillespie and J. Wyke (The Beatson Institute, Glasgow, United Kingdom). The pAll provirus is aBratislava strain (B77) of RSV cloned from an infected rat cell line and reconstructed with a 130-bp deletion between twoHindlll sites withinpol(19). Toestablish long-term

packaging

lines expressing thisvector,10 ,ugofpAllwascotransfected with 1 ,ug of pY3 containing the hygromycin resistance gene (hphr) (4)onto106Q2bn cells.hphrpopulationswereisolated andscreened forvirus release.

pASrcl (Fig. 3). A cloned EcoRI b fragment (containing v-src) from wild-type PrA RSV (kindly supplied by V. Fincham and J. Wyke [12]) was exchanged for the EcoRI fragment in pRAVKpn3' containing 3' env to 5' LTR se-quences. The resulting plasmid pRAVsrcl was used to constructthecomplete proviruspresentinpASrcl. Unique, internalBamHI, Sall,andHindlIl cloning sites arepresent inthis vector,as aresplicesites for thev-srcmRNA(RAV-1 donor, PrAacceptor).

pASrcneol (Fig. 3).ProviruspASrcneolwasderived from pASrcl. A BamHI-SalI fragment containing the neo gene and TK promoter from pIPB1 (a plasmid containing the neomycinresistance (neo) geneof TN5 (16) preceded by the herpes simplex virus thymidine kinase gene promoter (23), kindly given by R. Sweet (College ofPhysicians and Sur-geons, Columbia University, New York) was inserted into

the

BamHIISalI

cloning sitein pASrcl in thesame

transcrip-tionalorientationas v-src.

pASrcneo6 (Fig. 3). Provirus pASrcneo6 was derived from pASrcneol.Thethymidinekinase promoterwasremovedas

aBamHI-BglII fragment, and the plasmidwas self-ligated. The neo geneisexpresseddirectly fromthe viralLTR.

pASrcneo7. Provirus pASrcneo7 is similarto pASrcneo6, exceptthat the RSV EcoRI bfragment is derivedfrom B77 RSV and notPrARSV.

DNA transfection anddrug selection. Calcium phosphate-mediated DNA transfection was performed essentially as describedbyGraham and Van der Eb(15).Precipitateswere left on cells for 4 to 6 h, and the cells were then glycerol shocked(completemediumplus15%glycerolfor 100s).To establish stableQT6linesexpressing

pRAV-1*-,

the follow-ing DNAs weretransfected (per 106cells): 1 ,ug of BamHI cut

pIPBj;

10 ,ug of Sall-cleaved

pRAV-lq-;

10 ,ug of calf thymus DNA carrier. These cells were split 1 day after transfection and placed under G418 selection (200 to 400 jig/ml), and resistant colonies were isolated 2 to 3 weeks later. A second series of

RAV-1--expressing QT6

lines weredevelopedinasimilarmannerwith thehphrexpression plasmid PY3 (4)asthecotransfected marker.

Plasmids containingv-srcexpression vectors were trans-fecteduncut(10 ,ug)with 10 ,ugofcalfthymusDNAcarrier per 106 cells.

Virus collection and characterization withexpressionfocus unit (EFU) assay. Viruseswere collected from cells in fresh medium for 2.5 h. These supernatants were cleared of cell debris by centrifugation (5 min, 600 x gto 1,000 x g) and either used directlytoinfect cellsorstored frozenat-70°C before use. After transfection of v-src expression vector DNA into packaging cells, virus was collected 18 to 24 h posttransfection asdescribed above.

An immunocytochemical assay has been specifically de-veloped forquantitationof bothreplicatingand rdinfectious viruses (36). Briefly, CEF were infected with virus and

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TABLE 1. pRAV-LTR-andpRAV-1%--derived virus titers releasedfrom transfected and infected CEF andquailcell lines

Maximal virus Cell type Virusexpressed (titer(EFU/ml)

CEF NONE <1

CEF RAV-LTRa 1.0 x 106

CEF RAV-lh-a <1

QT6 RAV-LTRb 1.3 x 105

QT6

RAV-lq-F

line Qlan <1

QT6 RAV-1lt-line Q2an <1

QT6 RAV-ltl- lineQ2bn <1

aViral DNA wasintroduced by transfection, and virus titers were assayed

8days later after one cell passage.

bRAV LTRwasintroducedby infection, and virus titers were measured

after 12 days.

IViral DNA was introduced by transfection, and virus titers were assayed

at numerousintervals up to the present.

overlaid withagar 14 to 16 hlater. The agar was removed 5 to 10 days later, andthecells werefixed in2% paraformal-dehyde. A three-layer immunodetection system was em-ployed: layerone was an antiviral serum(either

anti-pl9g'g,

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two different monoclonal antibodies kindly provided by T. Pawson [MountSinaiHospital Research Institute, Toronto, Ontario, Canada] and D. Boettiger [University of Pennsyl-vania, School of Medicine, Philadelphia], or anti-pp60vsr( [JB327] kindly supplied by J. Brugge [State University of New York at Stony Brook, School of Medicine, Stony Brook]); layer two was a biotinylated anti-mouse F(ab')2 (Amersham Corp.); layer three was a streptavidin-conju-gated alkaline phosphatase complex (Enzo Biochem or Zymed).Aftersequential binding of these threereagents, the color reaction was developed to reveal foci of antigen-expressing cells (virus titers are expressed in EFU). For characterization of nonreplicating virus, the agar overlay step was omitted, and cells were stained 3 to 5 days postinfection. Thisassayishighly sensitive, detectingasfew as oneinfectioneventper testplate. Inaddition, ASrcneol-andASrcneo6-derived viruseswere quantitated bycounting the number ofQT6 neomycin-resistant (neor) colonies re-maining after virus infection andG418 selection (200 to 400

jig/ml).

RESULTS

Introduction of L-region deletion in cloned helper virus genome.

Analysis

ofnaturally occurring packaging-deficient RSV mutants suggested that critical RNA packaging se-quenceslie intheviral Lregion. We used suitablerestriction sites located in a cloned helper virus genome pRAV-LTR (containingcombined sequences fromRAV-1 andAEV10), to removepart ofL;adeletion ofapproximately150bpwas introducedbetween theBstEIIsite within theprimer-binding site(PBS)abutting the5' LTRandthe downstream Saclsite in L (Fig. 1and2) (see MaterialsandMethods). Inaddition to deleting a part of L, this removed 10 of the

original

18 nucleotides from the PBS. Because the L

region

is

dupli-cated in the downstream permuted RAV-1 sequences, this region and approximately 100 bp of 3' adjoining sequence were also removed to avoid recombination. The final pro-viral construct was named

pRAV-1V-.

RAV-1*-

is replication defective. To test whether the deletionmadein

pRAV-1*-

had removedsequences critical for virusreplication,wetransfectedcircular

pRAV-ltf-

and pRAV-LTR DNAs into CEF and monitored viral protein

expression

by

immunocytochemistry

and virus release.

Within8 days of transfection with pRAV-LTR, virus spread had occurredtomostcellsasjudged by expression of p199a9 (data not shown). High titers of infectious virus were re-leased from these cultured cells(Table 1). CEF transfected with

pRAV-1lq-,

however, did not show this pattern. Rather, a minor population of CEF (<0.01%) showedp199a9 expression, demonstrating stable transfection of RAV-1-in a small numberof cells without any indication of virus spreadorinfectious virusrelease (Table 1; seeFig. 5f).

To further characterize the behavior of pRAV-1l-, we transfected thecloned provirus intoQT6cells. As with quail cells in general, this tumor cell line has no endogenous viruses closely related to ASLV (13), although distantly related sequences have recently been described (5). The absence of endogenous viruses in QT6 is advantageous if recombination of introduced viral sequences (here

pRAV-1*-)

withcellular

t4

sequences istobeavoided. The pRAV-1- DNAwascotransfected into QT6 cells withaselectable neorplasmid,and neorcolonieswereisolated, amplified,and immunocytochemically stained for p199a9 expression. Three positive lines, Qlan, Q2an, and Q2bn, were chosen for detailed study.

Usingthe EFU virusassay (see Materials and Methods), we tested whether these lines released replicating helper virus and found none (Table 1). Moreover the lines have been nonproducers for more than 9 months and are thus stable. QT6 cells infected with the pRAV-LTR-derived helper virus produced high titers ofreplicating virus(Table

1).

Intact proviral DNA has been detected in each line by

TRANSfECTED

18-24 HR LATER COLLECT VIRUS IN SUPERNATANT

ASrcneol

I

INFECT 0T6 CELLS WITH VIRUS

I

G418 SELECTION

t

COUNT G418r COLONIES

All, ASrcl, ASrcneol

INFECT CEF WITH VIRUS

I

3-4 DAYS

(2) (1)

COLLECT

SUPERNATANT

INFECT CEF

3

3 OAYS FIX AND

IMMUNOSTAIN

FIG. 4. Regimen for testing rd virus packaging in RAV-lF--expressing QT6 cell lines. QT6* indicates QT6-derived packaging

cells.Supernatantswerecollected fromCEF(step 1)beforefixation ofthesamecells(step 2). Methodsfortransfection,viruscollection, andimmunostainingaredescribed inMaterials and Methods.

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a

c

b

d

af

f

FIG. 5. Alkaline phosphatase-immunostained foci of virus-in-fected CEFexpressing pp6-src (dark cells)in anuninfectedCEF

background. Panels: representative foci of cells 3 to4 daysafter

infection with virus derived frompAll (aandb), pASrcl (c), and pASrcneol (d), uninfected CEF 4 days after inoculation with

supernatant from pAll-transfected QT6 cells, stained with anti-pp6Ov-src (negative control, e); CEFexpressingpRAV-lp- 8days after transfection andasinglepassage, stained withanti-p199'9 (f). Bar,0.1mm.

Southern analysis of restriction endonuclease-digested cel-lular genomic DNA(data not shown). This rules outgross

proviral rearrangements as the reason for the absence of virus production by these lines. The lack of

RAV-1*-replication in QT6 cells corroborates the CEF transfection data discussed above, confirming that the deletion in RAV-1l- eliminated its ability to releasereplicating virus, whereas viral p199ag expressionwas maintained.

RAV-14--expressingcellscanpackage replication-defective

vectors. (i) Packaging ofpol- rd RSV. To test whether the mutationinRAV-lW-wascisacting,wetested whetherthe expressing QT6 cell lines couldpackage rd viral RNA into infectious viruses without accompanying helper virus. The first rdvectorwe testedwaspAll, apol- Bratislava strain

(B77) RSV provirus (19). Uncut pAll DNA (10 ,g) was

transfected into the lines Qlan, Q2an, andQ2bn, and tran-sient virusreleaseinto culturesupernatantswastested 18to 24 hlater (Fig. 4; Fig. 5a and b). Traditional focus assays were not performed because we have shown that our

im-munocytochemical assay is more accurate (36; A. Stoker,

unpublished

observations). pAll-derived

viruswasreleased from linesQlanandQ2bn, butnone wasdetected from line Q2anorQT6 cells

(Table

2).Thefact that

Q2an

cells didnot release virus

suggested

that the

provirus(es)

in these cells were debilitated through mutations or rearrangements not detectable

by

Southern

analysis,

and this linewastherefore notstudiedfurther.

To examine whether

replicating

viruswas

concomitantly

released withrd

virus,

supernatantsfrom the infected CEF were collected before cell fixation and used to infect fresh CEF

(Fig. 4).

These cells werelater fixed and

analyzed

for

p199a9

EFU.

Using

RAV-LTR

helper

virusweconfirmed the

sensitivity

of this

procedure: single plates

of CEF infected with1 to2 EFU of

replicating helper

virus released detect-ableprogeny virus

(up

to 10

EFU/ml)

into the supernatants 3

days

after infection.In

testing

the

pAll-derived virus,

two ofnine

independent experiments

revealed recombinant in-fectious virus in supernatants from infected CEF.

However,

virus titerswere verylow(12 and1

EFU/ml, respectively),

and the virus was

transforming

as

judged by positive

pp60v-src staining

and the transformed

morphology

of in-fected CEF. These recombinant viruses appear to have arisen

predominantly by

recovery of the pol sequence

by

All.

(ii)

Packaging

of vectors with no viral structural genes. From the above datawe could conclude that the cell lines

provided

pol functions and

expressed

gag

proteins.

We wished to ascertain whether

pRAV-l1--expressing

cells would

provide

all viral structural

proteins

in trans, andalso whether

by

incorporating larger

deletions into vectors the recombination with

RAV-1b-

would be reduced. Two smallerv-src vectors wereconstructed which lackedall viral structuralgenes.

pASrcl

retains

complete

5'

*

sequences in

L,

the 3'

polypurine

tract

(35),

viral

splice signals

for v-src

mRNA,

and thev-srcgeneofPrARSV.

Only

truncatedgag andenv

sequences remain of the viral structural genes. Transient virus releaseassayswere

performed

asdescribed abovefor

pAll.

pASrcl-derived

virus was released from both cell lines

tested,

attiters

comparable

to those found with

pAll

(Table 2).

pASrcneol

expresses bothv-srcand thedominant select-able neo gene

(under

the internal control ofthe HSV TK gene

promoter)

(Fig. 3).

The titer of

pASrcneol-derived

virus can be estimated from the number of neor colonies induced in infected

QT6 cells;

transient

expression

of

pASrcneol

in

Q2bn

cells thus gave maximal virus titers of 5.1 x 103 EFU/ml. ASrcneol virus was also

packaged by

line

Qlan,

butatlower

efficiency

than

Q2bn

(Table

2).

CEF infectedwith this rd viruswereimmunostained for

pp6Ovsrc

and foci with similar characteristics to those induced

by

ASrcl were observed

(Fig.

5c and

d).

The virus titers estimatedfromneor

colony

formationwere

broadly

compa-rabletothe

pp6Ov-src EFU, indicating

thatboth

quantitation

procedures

have similar accuracy.

Replicating helper

virus was not detected when

pASrcl

was tested in transient assays.

Similarly,

no

replicating

helper

was found when

pASrcneol

was

tested,

with one

exception: helper

virus wasfound afterone transfection of

Q2bn cells,

but

only

at the threshold of our detection

technique

(1 to 2

EFU/ml).

Helper

release was not

stable,

however,

since a later virus collection failed to detect its presence.

Long-termvirus-producinglines. The virus

analyses

using

thetransientassay demonstrated the

packaging

behavior of

RAV-1b--expressing

cell

lines,

but thetitersobtainedwere J.VIROL.

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TABLE 2. Transient vector-derived virus release from quail cell lines

Celltype Titer (EFU/ml) with the following rd vectorintroduceda: colonies/ml'

transfected pAll pASrcl pASrcneol

QT6 <1 (<1) <1 (<1) <1(<1) <1

<1 (<1) <1 (<1) <1(<1) <1

Qlan 3.0 x 102(<1) 5.5 x 102 (<1) 1.8 x 102(<1) 1.1 x 102

4.0 x 102(<1) 6.6 x 102(<1) 1.4 x 102 (<1) 1.4 x 102

7.4 x 102(<1) 2.0 x 102(<1) 1.2 x 102 (<1) 3.1 x 102

1.5 x 103(<1)

Q2bn 9.2 x 102(<1) 9.6 x 102(<1) 3.8 x 102(<1) 3.5 x 102

1.0 X 103(12) 1.0 x 103(<1) 3.4 x 103(1-2) 5.0 x 103

1.5 x 103(<1) 2.8 x 103(<1) 6.0 x 102 (<1) 5.1 x 103

1.8 x 103(<1) 3.5x103(1 )

Q2an <1 (NT)

<1(NT)

a Titer of virusreleased measuredwithpp6Ov-srcimmunostaining inCEF.Figures within parenthesesrepresenttiters ofreplicatingvirus releasedfrom infected CEF, measured in pl9gagEFU permilliliter. NT, Nottested.

bpASrcneol-derived viruswasanalyzed.

not satisfactory foruse in in vivo studies. In an attempt to

obtain higher titers, stable virus-releasing clones of cells

were isolated. Two approaches were used. In the first

approach we cotransfected pAll and an hph selectable

plasmid into line Q2bn and isolated stable hphr cells. These cells released pAll-derived virus at titers up to 30-fold

greaterthan the comparable transient titers (Table 3). Rep-licating viruses (including nontransforming helper virus)

were also detected at low levels with this large vector.

Although certain populations released as few as 0.01%

replicating viruses, we have not been able to subclone any

which were completely helper free, indicating that a

rela-tively high recombination rate was occurring between this

vectorand RAV-14- (either beforeor after virus release).

Our secondapproach wasto establishpackaging popula-tions by direct selection for transfected neo vectors. For this, wefirstestablishedasecond series ofpackaging lines,

using hph cotransfection with pRAV-1l-, which behaved essentially like Qlan and Q2bn described above in transient virus releaseassays (datanotshown). One such line, Q4dh,

wastransfected with pASrcneo6 and selected for neor (neo

and hph are independently selectable [4]). pASrcneo6 ex-presses neo directly from the viral LTR (Fig. 3), since we

wished to select for LTR activity and not an independent

internal promoter (pASrcneo6 gave titers comparable to

those ofpASrcneolin transientassaysin lineQ2bn). neor populations cloned in this way stably released ASrcneo6 virusattitersgreaterthan 105 EFU/ml (Table 3). Virus stocks were screened for replicating viruses as

de-scribed for Asrneol in Fig. 3. Virus screens up to7 weeks after clone isolation did not not reveal any helper virus.

Tests after 9 weeks did detecthelper virus, onlyatthe lowest threshold ofourdetection(Table 3). Other stable lines have

been isolated with vectorpASrcneo7, which is structurally similar to pASrcneo6; again, maximal rd virus titers were

greaterthan 105 EFU/ml. The majority of these lines have remainedhelper free since isolation (>10 weeks) (Table 3).

DISCUSSION

Our goals in this work were twofold: (i) to design and

express, inappropriate cells, acrippled ASLV-based helper

virus which would complement rd vectors in trans and yet withhold its own ability to replicate, and (ii) to design specific rd vectors for use in these packaging cells. In accomplishingthesegoals,wehave used novel immunocyto-chemical techniques to screen for replicating virus and infectious rd viruses.

The defective helper virus pRAV-14- was successfully expressed aftertransfection into QT6 cells, and these lines did not release replicating helper virus. The helper-free stability of the cells, an important consideration in their long-term use, may arise from the absence ofendogenous viruses available for recombination. We have also shown that stable expression ofpRAV-1t- occurs in CEF after transfection, without detectable virus replication (Fig. 5f; Table 1). These latter data are at odds with thosepresented by Cooper and Okenquist, who suggested that stable inte-gration of transfected rd viral DNA in CEF requires first virus replication and then reinfection (8). Our data with

[image:6.612.318.560.529.679.2]

pRAV-l1i-

argueagainstthisinterpretationastheexclusive

TABLE 3. Virus titers from stablevirus-releasing packaging cells

Cell Vector Virus- Virustiter Replicating

line expressed cell clonesa (EFU/ml) virus/ml

Q2bn pAll 1 2 x

104b

1-5

2 3 x 104 1-5

Q4dh pASrcneo6 1 2 x 105c 1-2

2 2 x 105 1-3

3 2x105 1-3

4 3 x 104 NTd

5 3 x 104 NT

6 6 x 103 NT

Q4dh pASrcneo7 1 2 x 105 <1

2 3 x 105 1-4

3 2x104 <1

aPopulationsisolated from colonies after transfection ofpackagingline and

drug selection.

bVirustitermeasuredbyp199agEFU.

cVirus titer measuredbyneorcolonyformation.

dNT,Nottested.

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route of stable expression; by immunocytochemistry we detect stable pRAV-l+- expression in CEF populations 8 days posttransfection. This form of expression after trans-fection into CEF is clearly very low, however, and differen-tial sensitivity of assay techniques may explain the differ-ences between ourdata and those ofCooper andOkenquist. Until recently the characterization of the 5' RNA packag-ing signals in ASLV genomes had not been extensive. The construction ofpRAV-li- was therefore foundedupon data from two naturally occurring RSV packaging mutants (27, 34).We have shownthat the deletion in pRAV-1l-, extend-ing from within the PBS region to a Sacl site in L, is sufficient to prevent the release of replicating virus from avian cells and that this defect is cis acting. While pRAV-lt-was being tested in our laboratory, Katz et al. presented adetailed analysis of theijregion in RSV, concluding that a critical30-bppackaging sequenceliesupstream ofthesingle SacI site in L (17). This 30-bp sequence is absent in pRAV-1-,and ourdata thus corroborate those of Katz and co-workers. When compared with TK15, however, the re-moval of L sequences does not satisfactorily explain the total lack ofpRAV-l+- replication. The mutant TK15 con-tains adeletion extending from the 3' junction of the PBSto near the gag AUG start codon (28). Although this region is about 100 bp larger than that deleted in

pRAV-1*-,

low-titer infectious virus is released fromTK15-expressingcells (18). Acritical difference between TK15 andRAV-1l-islikely to bethe removal of PBS sequences in the latter. A study by Pugasch and Stacey (30) showed that removal of the entire PBS from avian leukosis virus eliminated virus replication, presumably through blocking minus-strand DNA priming (for a review, see reference 38). The pRAV-l1i- genome retains only 8 of the original 18 bases of the PBS (additional base matches may occur with the newly juxtaposed sequence), and this may also be insufficient for specific tRNA binding. Although we have no biochemical data, we believe primarily that the deletion in L severely reducesor abolishes the specific packagability ofRAV-1- RNA, and that the PBS deletion prevents reverse transcription of any nonspecifically packaged viral RNA.

We have shown that pRAV-l+- linescanpackagea pol-B77 RSV and two specifically designed rd v-src vectors in transient assays. Significant recombination between vector pAll and

RAV-1*-

was shown to occur in these assays, and the evolution of replicating viruses in stable All-releasing populationsconfirmed this finding. WithvectorspASrcl and pASrcneol, replicating viruses were not detected in tran-sient assays exceptfor the single casediscussedabove. The stable lines releasing packaged vectors ASrcneo6 and ASrcneo7 also demonstrated that high titers of rd viruscould beisolated from

pRAV-lip--expressing

cells, with very low orundetectablerelease of replicating virus. Thus removalof unnecessary coding sequences in the pAS series of vectors significantly reduces recombination frequencies in compari-son to pAll.

Although the removal of internal sequences also generates space for nonviral inserts, maximizing the size of the dele-tion must be balanced against the retention of necessary cis-acting sequences. Recent studies of both avian and murine retroviruses indicate that sequences in the 5' gag region may augment virus expression and/or genomic RNA encapsidation and also influence the viral disease spectrum (1, 2, 33). RSV hasbeen shown tocontainasequencein gag with transcriptional enhancing activity and also a sequence flanking the BamHI site at position 532 (PrC RSV base equivalent) which may enhance encapsidation (32). Our pAS

vectors,however, donotcontain these regions. Removal of the gag enhancer segment appears not to hinder vector expression as judged by the ability of the vector-derived viruses to morphologically transform CEF (A. Stoker, un-published observations). Nevertheless, we are presently examining thisquestionin moredetail. Loss of theputative encapsidation region does not significantly affect the virus titers derived from our vectors in comparison with either pAll (in both transient and long-term virusrelease;Table3), or the titer of RAV-LTR released from QT6 (Table 1). Therefore RNApackagingremains efficient after loss of this region, afindingalso notedbyothers(20, 28).

Wehave shown withimmunocytochemistrythataccurate quantitation of vector-derived viruses is possible without using internal selectable markers. Quail cellsarealso resis-tant toinfection by a number of commonASLV subgroups (25),and therefore the useofthe neor

colony-forming

assay for such viruses would not be applicable. If immunocyto-chemical detection isafeasible

alternative,

selectablevector genes may be either removed or replaced by sequences of biological interest in size-constrainedvectors.

In conclusion, we have constructed an ASLV-based helper virus derivativewithacis-acting mutationpreventing the release ofreplicating virus. Stable quail cell lines ex-pressing pRAV-l+- are helper virus nonproducers but are able to package rd ASLV-based vectors into infectious virus. Using cell clones selected tostablyexpressrdvectors, we have obtained high-titer rd virus with very low or undetectable helper contamination, suitable foruseinvivo. Theavailability of this cell systemwill furtherour examina-tion ofthe complex oncogene-host interactions in hatched andembryonic avian tissues. Moreover, suchasystem will facilitate experiments in vivo

addressing

broader

questions

ofviralexpression, target

specificity,

and cell

lineage

in the developing avian embryo.

ACKNOWLEDGMENTS

We thank Jill Hatier for preparation of CEF and the persons mentioned in thetextforprovisionofantibodies andplasmids. We thankL.-H.Chen,M. Glotzer,A. R.Howlett, andR.Schwartz for helpfulcommentsconcerningthemanuscript.

This work wasfunded inpartby aEuropean MolecularBiology Organization fellowship toA.W.S. (ALTF 271-1985), and by the Office of Health and Environmental Research, Department of En-ergy,undercontract DE-AC-03-76SF00098.

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