JOURNAL OF VIROLOGY, Mar. 1979, p. 1023-10340022-538X/79/03-1023/12$02.00/0

Vol. 29, No. 3

Isolation and Characterization of a Mouse Cell LineContaining a Defective Moloney Murine Leukemia Virus

GenomePETER BESMER,1 HUNG FAN,2 MICHAEL PASKIND,1 AND DAVID BALTIMORE'*

Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139,1 and Tumor Virology Laboratory, The Salk Institute, San Diego, California 921122

Received for publication 13 September 1978

A culture of mouse cells containing a 1,000-nucleotide deletion mutant ofMoloney murine leukemia virus has been isolated. The deletion did not affect thesize or function of the 21S mRNA that encodes the env gene products. Both thedeleted RNA and the 21S mRNA were recovered in polyribosomes. Cells con-

taining the deleted virus made no detectable Pr1809'". Pr659'9 synthesis wasalso absent, but a 45,000-molecular-weight gag gene product was found thatmight be encoded by the deleted genome. Biosynthesis of Pr80env proceedednormally in these cells; the intracellular precursor was cleaved and migrated tothe cell surface as gp7O. The cells could not be superinfected by homologousMoloney murine leukemia virus presumably because of surface restriction due tothe gp7O. Although the cells express the Moloney murine leukemia virus gp7O on

their surface, they will not make pseudotypes after infection with vesicularstomatitis virus implying that Pr659'9 may play a critical role in pseudotypeformation. Induction of endogenous virus expression in the cells carrying thedeletion mutant generated an N-tropic murine leukemia virus that can fuse XCcells. This may represent a recombinant between the deletion mutant and an

endogenous virus.

Although infections ofmouse cells by a murineleukemia virus (MuLV) are usually thought toresult in a virus-producing infected cell, thereare reports of nonproductive cells that produceviral antigens and were therefore infected (42,47). Such a state is also seen in cultured normalcells from certain mouse strains and reflectsexpression of endogenous viral genomes; in fact,

most mouse cells express some viral antigen butmake no virus (6, 32, 38, 51). __-A few years ago we noted that a culture of

cells infected with Moloney MuLV (M-MuLV)was producing decreasing yields of virus withcontinuous passage. We have recovered fromthat culture a nonproducer cell line that ex-presses viral glycoprotein but little of the otherviral proteins. Cells productively infected withM-MuLV make gene products from the threeknown genetic regions of the M-MuLV genome:gag (the internal virion proteins), pol (the re-verse transcriptase), and env (the glycoprotein).The nonproducer cell line isolated in these stud-ies is infected with a deleted M-MuLV genomethat makes no normal gag or pol products buthas an apparently normal env gene product anda normal env mRNA.

MATERIALS AND METHODS

Cells and leukemia viruses. Most of the cells andviruses have been described previously. JLS-V11 cells(58) were provided by Electronucleonics Inc., Be-thesda, Md., 18 passages after their infection with M-MuLV. JLS-V9 (V9) cells (58) were obtained fromKenneth Manly (Roswell Park Memorial Institute).NIH/3T3 cells (26) were a gift of S. A. Aaronson(National Institutes of Health). BALB/3T3 cells cloneA31 (1) were obtained from R. Pollack, and the 8csubclone 81 cat cells, a sarcoma-positive leukemia-negative (S+L-) cat cell line transformed by Moloneymurine sarcoma virus (17), was kindly provided by P.Fischinger. NIH/3T3 cells producing M-MuLV clone1 virus were derived in this laboratory (16). XC cells(52) were provided by J. Hartley (National Institutesof Health). Cells were grown in Dulbecco-modifiedEagle medium with 10% calfserum (NIH/3T3, BALB/3T3, XC, and NIH cells producing M-MuLV) or with10% heat-inactivated fetal calf serum (JLS-V9, JLS-Vli, and all subclones derived from JLS-V11). Thecat S+L- cells were grown in McCoy SA medium plus14% heat-inactivated fetal calf serum.M-MuLV infections and direct plaque assay.

Plaque assays were performed with the XC cell fusionprocedure (44). For quantitation of MuLVx (xeno-tropic MuLV), the focus assay described by Fischingeret al. (17) was used. Reverse transcriptase assays of

1023

1024 BESMER ET AL.

supernatants were done as described elsewhere (20).Infective center assays were carried out by a modi-

fication of a published procedure (26). SensitiveBALB/3T3 cells were plated at 105 cells per 6-cm dish.The next day, cells to be tested were trypsinized for 10min with 0.5% trypsin at room temperature, dilutedinto medium, and then plated onto the indicator cellsat various densities. After 3 days the cultures were UVirradiated and overlaid with XC cells. The platingefficiency of the cells was determined separately. JLS-V9 and JLS-V11 routinely gave plating efficiencies of60 to 80%.

Antisera. Fluorescein-conjugated antibody toTween-ether-disrupted M-MuLV made in goats andrhodamine counter stain were obtained through theVirus Cancer Program. Anti-Friend MuLV gp7O serummade in rabbits was kindly provided by D. Bolognesi.Goat anti-rabbit immunoglobulin and normal rabbitserum were purchased from Meloy Laboratories. Anti-MuLV p30 serum made in rabbits was kindly providedby David Livingston. Anti-M-MuLV p30 and gp69/70antisera made in rabbits were prepared in this labo-ratory (39).Radioimmunoassays. The cell extracts were pre-

pared as follows. Subconfluent plates of cells (15 cm)were harvested with phosphate-buffered saline (PBS)(pH 7.4)-2 mM EDTA and then washed three timeswith PBS. Cells were stored as 20% cell packs at-70°C. The cell packs were disrupted in 1% Triton X-100 and 0.5% sodium deoxycholate, and the nucleiwere collected by centrifugation at 2,000 rpm for 4min. The supernatant was then extracted twice withether, and the aqueous phase was dialyzed with 0.05M Tris-hydrochloride (pH 7.8) and used for the ra-dioimmunoassay. The radioimmunoprecipitation as-says for the p30 protein were performed as describedby Parks et al. (38). Protein determinations were doneby the Lowry (31) method with bovine serum albuminas a standard. The first incubation was carried out in450 td of 0.1 M NaPO4, (pH 7.5), 1% normal rabbitserum, 0.01% Triton X-100, 0.01 M EDTA, competingantigen and rabbit anti-M-MuLV p30 for 1 h at 37°C,and then 50 pl of '2I-p30 (10,000 cpm) in 0.1 M NaPO4(pH 7.5), 0.01% Triton X-100, 0.01 M EDTA, and 1%normal rabbit serum was added and incubated foranother hour at 37°C. Finally, 30 ul of goat anti-rabbitimmunoglobin serum was added, and the mixture wasincubated for 1 h at 37°C. The precipitate was col-lected by centrifugation for 10 min at 3,000 cpm. Thesupematant was decanted, and the pellet was washedwith 0.5 ml of 1-mg/ml bovine serum albumin-0.1%Nonidet P-40 in PBS. The precipitate was again col-lected by centrifugation. This washing procedure wasrepeated three times. The tubes were then counted ina gamma-ray counter.Immunofluorescence. Cytoplasmic staining was

carried out as described previously (20).Membrane staining of live cells. The procedures

for staining antigens on the surface of live cells wereprovided by Nancy Hopkins (unpublished data). Coverslips with cells were washed five times in PBS con-taining 1 mM CaCl2 and 0.5 mM MgCl2 (PBS-MC). A25-,ul amount of a 1/40 dilution of fluorescein-conju-gated anti-M-MuLV antibody and a 1/100 dilution ofrhodamine counterstain in PBS-MC was added, and

the cover slips were incubated for 30 min in a moistchamber at 37°C. They were then washed five timesin PBS-MC and mounted onto slides with PBS-MCand 50% glycerol.

For membrane staining of formaldehyde-fixed cells,cover slips with cells were washed five times in PBSand then fixed for 20 min in PBS containing 3.5%formaldehyde at room temperature. The cover slipswere then washed five times in PBS and stained asdescribed in the procedure for acetone-fixed cells.The method used for indirect staining with noncon-

jugated rabbit anti-glycoprotein antiserum was as fol-lows. For membrane staining of live cells, cover slipswith cells were washed with PBS-MC, 25 pi of a 1/20dilution of anti-glycoprotein rabbit antiserum in PBS-MC were added, and incubation was carried out for 30min at 37°C in a moist chamber. The cover slips werethen washed five times in PBS-MC, 25 ul of a 1/40dilution of fluorescein-conjugated goat anti-rabbit an-tiserum and a 1/100 dilution of rhodamine counter-stain in PBS-MC was added, and the second incuba-tion was carried out for 30 min at 37°C. The coverslips were washed extensively and mounted as de-scribed above. For cytoplasmic staining of acetone-fixed cells and for membrane staining of formalde-hyde-fixed cells, the procedure was modified accord-ingly.Metabolic labeling and gel electrophoresis.

Cultures about 70% confluent (10-cm plates) werelabeled for 2 h with [35S]methionine (New EnglandNuclear Corp., 50,uCi/ml) in Dulbecco-modified Eaglemedium containing 1/50 the normal methionine con-centration and 10% dialyzed heat-inactivated fetal calfserum. The cells were lysed with 0.01 M phosphate(pH 7.2), 1% Triton X-100, 0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate (SDS), and 0.15 M NaCl.The lysates were scraped from the plates and clarifiedby centrifugation for 10 min at 1,000 x g.To reduce nonspecific background in the immuno-

precipitation, the lysates were precleared by the tech-nique of Witte et al. (56). Extracts were incubatedovernight with 10 pl of normal rabbit serum at 4°Cand then for 1 h at 4°C with 100 pl of 10% (wt/vol)Formalin-fixed Staphylococcus aureus as previouslydescribed (28). The components bound to S. aureuswere collected by centrifugation for 30 min at 35,000rpm in a Beckman ultracentrifuge.The supernatants were then immunoprecipitated

by overnight incubation at 4°C with the appropriateantiserum followed by 1 h of incubation with S. aureus(as above). The precipitates were washed three timeswith lysis buffer, resuspended in 50 ,ll of 50 mM Tris(pH 6.8)-1% 2-mercaptoethanol-1% SDS-10% glyc-erol-bromophenol blue, boiled for 5 min, and analyzedon 5 to 20% gradient slab gels (29). The gels werestained and dried, and the labeled bands were visual-ized by fluorography and autoradiography at -70°C(9).

Extraction and analysis of cytoplasmic RNA.Cytoplasmic RNA from producer and nonproducercells was prepared as described previously (14) and bythe urea-SDS method (19) as described by Sharp etal. (46). Polyadenylic acid-containing RNA was pre-pared by affinity chromatography on oligodeoxythy-midylic acid-cellulose (T3, Collaborative Research)

J. VEROL.

MOUSE CELL LINE WITH DEFECTIVE M-MuLV GENOME 1025

(3). Cytoplasmic RNAs were analyzed in 15 to 30%sucrose gradients containing 0.1% SDS as describedpreviously (14). Purified polyribosomes were preparedas described previously by sedimenting polyribosomesfrom cytoplasmic extracts through 1 and 2 M sucrose

(15, 18). Electrophoresis of RNA on denaturing meth-ylmercuric hydroxide agarose gels was performed as

described by Bailey and Davidson (4) with a slab gelapparatus (20 by 12 by 0.3 cm). Methylmercuric hy-droxide was obtained from Alfa Products (Danvers,Mass.) as a 1 M stock solution.

Preparation of M-MuLV cDNA and hybridiza-tion techniques. 3H-labeled M-MuLV complemen-tary (cDNA) was prepared by incubation of M-MuLVclone 1 virions (16) in vitro in the endogenous RNA-dependent DNA polymerase reaction, as describedpreviously (13). The specific activity of the M-MuLVcDNA was 1.6 x 107 cpm/jug as calculated from thespecific activity of the radioactive deoxyribonucleosidetriphosphate precursor. 3H-labeled M-MuLV cDNAthat had been depleted of uninfected BALB/c cell

complementary sequences was a kind gift of RudolfJaenisch, and its preparation is described by Jaenisch(23).

32P-labeled M-MuLV cDNA was prepared from M-MuLV grown in NRK cells (CP1 cells, E. Rothenberg,unpublished data) according to Taylor et al. (53) inthe presence of calf thymus oligodeoxynucleotideprimers. The initial specific activity of the cDNA was

108 to 2 x 108 cpm/,ug as calculated from the specificactivity of the labeled precursor.

Hybridization of cytoplasmic RNAs with M-MuLVcDNA to measure extent and concentration of virus-specific RNA sequences was as previously described(13). Briefly, the RNA samples were combined with3H-labeled M-MuLV cDNA (500 to 1,000 cpm) andincubated for the times indicated. Annealing condi-tions were 0.3 M NETES (0.3 M NaCl, 0.01 M TES,pH 7.5, 1 mM EDTA)-0.1% SDS at 66°C, and thetotal reaction volume was 5 to 7 p1. After annealing,the samples were expelled into 50 pl of S1 nucleasereagent (40 U of S1 nuclease per ml, 0.25 M K acetate[pH 4.5], 20 ,ug of denatured calf thymus DNA per ml,0.01 M ZnSO4), and the unhybridized cDNA was di-gested for 30 to 60 min at 45°C. The S1-resistantmatekial was precipitated with trichloroacetic acid,and radioactivity in precipitated material was meas-ured by filtration (Millipore Corp.) and liquid scintil-lation counting.

Hybridization across sucrose gradients was per-formed as described (13). Briefly, samples of sucrose

gradient fractions were adjusted to annealing condi-tions and M-MuLV cDNA (500 to 1,000 cpm) was

added to each sample. After incubation of all of thesamples for the same length of time, the hybridizedcDNA was measured and converted to relative virus-specific RNA concentration as described before (13).

Transfer of RNA from agarose gels to diazo-benzyloxymethyl paper. The aminobenzyloxyme-thyl paper was prepared from 1-[(m-nitrobenzyl-oxy)methyl]pyridium chloride and stored in a desic-cator at 5°C. Immediately before the transfer wasstarted the paper was diazotized at 4°C. The methyl-mercuric hydroxide agarose gel was quenched with 0.5M NH4Ac and stained with 10 ,ug of ethidium bromide

per ml. The gel was then placed into 200 ml of 50 mMNaOH-5 mM 2-mercaptoethanol and rocked gentlyfor 30 min at room temperature. The gel was thentreated with 200 ml of 200 mM potassium phosphate(pH 6.5) containing 7 mM iodoacetic acid for 10 minat room temperature and then twice with 200 ml of 25mM potassium phosphate (pH 6.5) for 5 min at roomtemperature. After treatment as above the procedurefor transfer was essentially the same as the one de-scribed by Southern (48) except that 25mM potassiumphosphate (pH 6.5) was used. Transfer was allowed toproceed overnight. After the transfer the paper waswashed twice in 200 ml of water, dried in air, andstored at 5°C until used for hybridization. For hybrid-ization to the RNA bound to the paper the procedurewas the same as described by Alwine et al. (2). Thepaper was pretreated overnight at 42°C with hybridi-zation buffer (50% formamide, 0.75M sodium chloride,75mM sodium citrate) containing 0.02% (wt/vol) eachof bovine serum albumin, Ficoll, and polyvinylpyroli-done; 1.5 mg of denatured calf thymus DNA per ml,and 1% (wt/vol) glycine. Hybridization was then per-formed in the same mixture without the glycine andthe probe (200,000 cpm/2 ml) for 36 h at 42°C. Thepaper was then washed with six to eight changes ofhybridization buffer at 37°C each for 30 min withrocking and then with three changes of 50 mM sodiumchloride-3 mM sodium citrate at 4°C. The paper wasblotted to remove excess solution, wrapped in SaranWrap, and visualized by autoradiography.

RESULTSJLS-V9 is a cell line that was derived from

BALB/c bone marrow; on infection with M-MuLV these cells were designated JLS-V1l (58).JLS-Vll cells were received in our laboratory atthe 18th transfer, and by the 40th transfer virusproduction had dropped markedly as measuredby the amount of reverse transcriptase activity,or particles banding at 1.16 g/cm3, in the me-dium. We were interested to determine whetherthis alteration in virus production was due to ageneral decrease in virus production in all cells,or whether it was the result of selection of virus-negative cells present in the original JLS-Vllculture. An infective center assay at passage 121revealed that only 0.4% of the cells were produc-ing M-MuLV, but the virus-positive cells pro-duced normal quantities of virus. Therefore, itappeared that we were dealing initially with aheterogenous population of cells, and thus wecloned early and late passage JLS-Vll cells.In a clone isolated from passage 21 of JLS-

Vll cells (hereafter referred to as "Vll" cells),all of the cells were producing virus as measuredby an infective center assay. After culture formore than 1 year (more than 50 transfers), theyremained stable producers. From the late trans-fer JLS-Vll cells (transfer 121), 15 clones wereisolated, all of which were negative for the pro-duction of type C virus both by release of plaque-

VOL. 29, 1979

1026 BESMER ET AL.

forming virus measured by XC cell assay and byparticle-associated DNA polymerase detectablein supernatants. The cells themselves also didnot induce syncytium formation when overlaidwith XC cells. When stained with fluorescentserum made against disrupted M-MuLV, all theclones stained strongly positive in the cytoplasmas well as on the cell membrane; the uninfectedJLS-V9 cells displayed no detectable fluores-cence. It appeared therefore that the nonpro-

ducer JLS-Vll cells (hereafter referred to as

"V11-NP") expressed viral antigens but were notable to produce any virus particles. The purpose

of the following experiments was to define thelesion in these cells.

Proteins. The genome of retroviruses codesfor three major polyprotein gene products: (i)Pr6Ssas, that is processed to give rise to most ofthe structural proteins of the virus (5, 45); (ii)Pr180909-P°', the precursor of p85 (reverse tran-scriptase) (25, 27, 36); and (iii) PrSOenv, the pre-

cursor of the envelope proteins, mainly the gly-coprotein gp7O (12, 34, 57). In characterizingVll-NP cells, we first determined whether any

of the viral proteins were expressed.The quantity of p30, the major structural vir-

ion protein, and a cleavage product of Pr65sagwas measured by radioimmunoassay (Fig. 1).V11-NP cells had no more p30 antigenic materialthan uninfected V9 cells (-10 ng/mg), whereasVll cells producing M-MuLV and V9 cells in-duced with bromodeoxyuridine to produce en-

dogenous MuLV (8) had high levels of p30 an-

tigen (500 to 1,500 ng/mg). In this radioimmu-noassay, iodinated Rauscher MuLV p30 and ho-mologous antibody made against Rauscher p30protein were used.

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To further investigate viral protein synthesis,polyacrylamide gel electrophoresis of specificimmunoprecipitates was used (Fig. 2). Extractsof [35S]methionine-labeled Vll cells (slots a tof, Fig. 2), V11-NP cells (slots g to 1), and V9 cells

(slots m to p) were analyzed with a variety ofantisera. V9 cells had no specific proteins. Vllcells showed the array of proteins previouslycharacterized in other systems: Pr1809)a9POI pre-

cipitated with anti-reverse transcriptase (slot a)and anti-p30 (slot c) and products from aboutp145 to p85 precipitated only with anti-reversetranscriptase; Pr65sas, p30, and some intermedi-ates precipitated with anti-p30 and anti-wholevirus serum (slot e); Pr80env precipitated withanti-gp7O (slot b) and anti-whole virus serum.

Some Pr65i'9 precipitated with the anti-reversetranscriptase serum because a known contami-nating activity in the serum reacts with the p15component of Pr65as (56). Normal rabbit serumprecipitated no proteins (slot f), and p30 com-

peted out all precipitation with anti-p30 (slot d).V11-NP cells had no detectable Pri8O9 9P" or

Pr659"9 with the same sera (slots g and i) but didhave Pr80env (slot h). The anti-whole virus serum(slot h) precipitated a diffuse band at 70,000molecular weight that is presumed to be thecleaved form of glycoprotein, gp7O. Why theanti-gp7O detected so little of this polypeptide isnot known, but this antiserum has previouslybeen shown to be much more avid for Pr80eMGthan for gp7O (47).A minor component in V11-NP cells, P45, was

precipitated with anti-p30 (slot i) and anti-wholevirus serum (slot k). The reactivity with anti-p30 was competed out with p30 (slot j). A P45polypeptide was also evident in Vll cells ex-

232.3

gg Protein addedFIG. 1. p30 radioimmunoassays. Cell extracts from NP (0), JLS- V9 (A), bromodeoxyuridine-induced JLS-

V9 (U) and NP (L), and JLS- V11 (x) cells were prepared, and the radioimmunoassay was carried out as

described in the text.

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J. VIROL.

230I'll

MOUSE CELL LINE WITH DEFECTIVE M-MuLV GENOME

a b c d e f g h i I k I m n o p

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FIG. 2. Immunoprecipitation of viralproteins from Vll and VlI-NP cells. (a to f) Immunoprecipitation of[3SJmethionine-labeled Vll cell extracts with (a) anti-reverse transcriptase serum, (b) anti-glycoproteinserum, (c) anti-p30 serum, (d) anti-p30 serum in the presence of an excess ofpurified, p30protein, (e) rat anti-whole virus serum, and () normal rabbit serum. (g to 1) Immunoprecipitation of[5S]methionine-labeled Vll-NP cell extracts with (g) anti-reverse transcriptase serum, (h) anti-glycoprotein serum, (i) anti-p30 serum, (j)anti-p30 serum in the presence of an excess ofpurified, p30 protein, (k) rat anti-whole virus serum, and (1)normal rabbit serum. (m to p) Immunoprecipitation of[5S]methionine-labeled JLS- V9 cell extracts with (m)anti-reverse transcriptase serum, (n) anti-glycoprotein serum, (o) anti-p30 serum, and (p) rat anti-whole virusserum.

tracts (slots c and e). Its concentration was verymuch less than that of the other p30-relatedpolypeptides in Vll cells, and we are uncertainwhether it derives from the M-MuLV genome orfrom an endogenous or contaminating virus.From the immunoprecipitation data, Vll-NP

cells appear to contain the glycoprotein precur-sor as well as finished gp7O. Immunofluorescentstaining of live and acetone-fixed cells with mon-ospecific anti-gp7O antiserum showed that theglycoprotein was found both in the cytoplasm offixed cells and on the cell membrane of live cells(unpublished data).To investigate whether both gp70 and Pr80env

were localized on the outer cell membrane, Vll-NP cells were radioiodinated with lactoperoxi-dase as catalyst. Immunoprecipitation and poly-acrylamide gel electrophoresis showed that onlylabeled gp70 was precipitated with anti-gp70(Fig. 3, lane a) even though the antiserum usedwas more avid for Pr8Oenv than for gp70; neitheranti-p30 nor normal rabbit serum precipitatedany protein bands (lanes b and c). The size ofPr8Oenv is evident from Fig. 3, lane d. When thelabeled cells were trypsinized, 70 to 80% of la-beled gp70 was removed, indicating that most of

it is accessible to trypsinization (data notshown). Vll-NP cells appeared to produce bothgp7O and its precursor, Pr8Oenv; only the gp70,however, was found on the outer cellular mem-brane.Surface restriction. The virus-negative Vil-

NP cells were isolated from a virus-producingculture, suggesting that the cells would be re-sistant to superinfecting MuLV. Otherwise, V11-NP cells should have been superinfected withM-MuLV from producer cells in the originalJLS-V11 cultures. To clarify this point, the in-terference properties of the Vil-NP cells wereinvestigated. Vl1-NP cells and control V9 cellswere infected with M-MuLV at a multiplicity ofinfection of 1, and supernatants were collected2 and 5 days after infection. Vll-NP cells pro-duced about 1,000-fold less virus than V9 cells(Table 1). To examine whether this strong re-sistance to superinfection occurred at the cellsurface, the pseudotype virus, vesicular stoma-titis virus (VSV)(M-MuLV), was produced byinfecting M-MuLV-producing cells with heat-la-bile VSV tl-17 (7, 59). The stock was then heatedat 45°C for various times to kill VSV(VSV), andresidual pseudotypes were titered on Vll-NP

180K-

80K-65K-

45K-

30K-

VOL. 29, 1979 1027

1028 BESMER ET AL.

70K

FIG. 3. Surface iodination of gp7O on Vll-NPcells. Surface iodination of the Vll-NP cells was

essentially carried out as described by Hynes (22).

After the iodination was stopped, the cells were

washed three times with PBS. The cell pellets were

lysed with 0.5 ml of 0.1 M Tris (pH 6.8)-2 mMphen-

ylmethylsulfonylfluoride-15% glycerol-2% SDS. Im-

munoprecipitation of the cell extracts was carried out

with (a) anti-glycoprotein serum, (b) anti-p30 serum,

(c) normal rabbit serum; (d) immunoprecipitation of

a [365]methionine-labeled Vll cell extract was car-

ried out with anti-glycoprotein serum.

TABLE 1. Interference ofNP cells with M-MuLV"

Cell Yield of XC cell (PFU/ml per 106 test cells) at:type 2 days after infection 5 days after infection

JLS-V9 8.6 x 105 1.5 x 105Vll-NP 1.3 x 103 1.3 x 102

a Cells (5 x 106) were plated onto 5-cm dishes, andon the next day the cultures were infected with M-MuLV at a multiplicity of infection of 1 in the presenceof 8 jig of polybrene per ml. After 2 h of adsorption,the inoculum was removed, and the cells were washedtwo times with regular medium. After 2 and 5 days,24-h harvests were collected and the virus yield wasdetermined in an XC assay with NIH/3T3 cells asindicators.

cells and V9 cells. Fig. 4 shows that only 10-2.5 ofVSV(M-MuLV) that could infect V9 cells was

able to infect Vll-NP cells. Therefore, Vll-NPcells showed a marked surface restriction to-wards M-MuLV.Lack of pseudotype formation. To exam-

ine whether the gp7O on Vll-NP cells is able toparticipate in pseudotype virion formation, thecells were infected with VSV tl-17. M-MuLV-producing NIH/3T3 cells were superinfected

v/v0

Minutes inactivation at 450 CFIG. 4. Interference of NP cells with VSV(M-

MuLV). Pseudotypes of the VSV mutant tl-17 withthe coat of M-MuLV were prepared as previouslydescribed (7) and inactivated at 45°C for the indi-cated times. VSVplaque assays (49) were performedwith JLS-V9 (0) and NP (A) cells as indicator cells.V/Vo is the surviving fraction after heating.

TABLE 2. Lack ofpseudotype formation betweenVSV and M-MuLV in Vll-NP cellse

Surviving VSV fraction on indica-Cells for heated tor cells:VSV tl-17 stocks

JLS-V9 Vll-NP

M-MuLV-infected 10-3 1o-6NIH/3T3

Vll-NP 3.5 x 10-6 1.5 x 10-6a Subconfluent plates of NIH/3T3 cells producing

M-MuLV and Vll-NP cells were infected with VSVtl-17 at 32°C at a multiplicity of infection of 1 to 2.The virus was absorbed in a volume of 0.5 ml/5-cmdish. After 45 min of adsorption, the inoculum wasremoved, the cells were washed once with regularmedium, and then 5 ml of regular medium was added.After 12 h of infection at 320C, the virus yield washarvested, and portions were heat inactivated at 45°Cfor 60 min. The residual fraction of VSV was thendetermined in a VSV plaque assay on JLS-V9 andVll-NP cells at 320C.

J. VIROL.

MOUSE CELL LINE WITH DEFECTIVE M-MuLV GENOME 1029

with VSV tl-17 in parallel, and the two VSVstocks were then heated at 45°C to eliminate thepure VSV in the populations. To detect pseu-dotypes, the stocks were assayed on V9 cells orVll-NP cells (Table 2). Although the stockgrown on the M-MuLV-infected cells had 103PFU of pseudotype VSV per ml that appearedon V9 cells but not Vll-NP cells, no such pseu-dotypes were evident in the VSV preparationmade on V11-NP cells. The gp7O on the surfaceof Vll-NP cells thus appears incapable of par-ticipating in pseudotype virion synthesis.Expression of M-MuLV-specific RNA in

producer and nonproducer cells. The M-MuLV-specific RNA in Vll-NP cells was ex-amined to determine whether the lack of viralproteins in the nonproducer cells was due to analteration in the expression of virus-specificRNA. Cytoplasmic RNA extracted from pro-ducer and nonproducer cells was annealed withM-MuLV cDNA in conditions of virus-specificRNA excess (13). At saturation, RNA from theVll-NP cells could anneal M-MuLV cDNA tothe same (maximal) extent as RNA from theproducer cells (Fig. 5), indicating that essentiallyall virus-specific RNA sequences that could berecognized by the M-MuLV cDNA probe werepresent in the nonproducer cells. In annealingexperiments performed in this manner, the vi-rus-specific RNA concentration is inversely pro-portional to the product of RNA concentrationand annealing time that gives half-maximal an-nealing (the Crtl/2 value) (13). Comparison withthe Crtl/2 value for pure M-MuLV 70S RNA

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FIG. 5. Annealing of cytoplasmic RNAs with M-MuLV-specific cDNA. Cytoplasmic RNA extractedfrom producer and non-producer JLS- VlI cells wasannealed with 3H-labeled M-MuLV-specific cDNAfor different lengths of time. The percent maximalcDNA hybridized is plotted as a function of the C,tvalue (product of RNA concentration and time ofannealing) corrected to standard conditions (10). TheCrtii2 values are also indicated. Symbols: 0, producerJLS- Vl I cell RNA; 0, nonproducer cell RNA.

(4.02 x 1o-2 mol-s/liter at standard annealingconditions [15]) indicated that 0.03% of the cy-toplasmic RNA from Vll-NP cells and 0.10% ofthe producer cell cytoplasmic RNA was virusspecific. The lack of virus production in thenonproducer cells, therefore, did not result frommarked alteration in the expression of virus-specific RNA because no change in extent of M-MuLV-specific sequences present was detectedand the concentration of these sequences wasonly threefold lower than for producer cells.

Uninfected BALB/c mouse cells contain RNAsequences that can anneal with M-MuLV cDNto a limited extent (6, 8, 14, It; at least some oftnese seuences may represent endogenous C-

type irsesTo ensure that tFig. 5 were analyzing transcription of M-MuLVcDNA and not uninfected cell sequences, pro-ducer and nonproducer cell RNAs were an-nealed with M-MuLV cDNA that had been de-pleted of sequences that anneal with uninfectedBALB/c cell DNA (a kind gift of Rudolf Jae-nisch). V11-NP and Vll cell RNA annealed toequal extents to the selected cDNA probe (Fig.6). The CrtI/2 values indicated that with theseRNA preparations, the nonproducer cell cyto-plasmic RNA contained approximately one-fourth as much virus-specific RNA as producercells (0.06 versus 0.26%).

Intracellular virus-specific RNA in cells pro-ducing MuLV consists of 38S and 21S virus-specific RNA (11, 13, 33, 43, 54). To examinewhether the same virus-specific RNA specieswere present in the nonproducer cells, cytoplas-

O-

nwN25CD

m>- 50-

zwcrwa.

100-

0O\ 65

16 6\

X \\ 0

0

0 0-

10 102 103Crt (MOL-SEC/LITER)

FIG. 6. Annealing of cytoplasmic RNAs with se-lected M-MuLV-specific cDNA. Cytoplasmic RNAfrom producer and nonproducer JLS-VII cells (dif-ferent preparations from Fig. 5) was annealed withM-MuLV cDNA which had been depleted ofBALBIc uninfected cell sequences. Analysis was the same asin Fig. 5. Symbols: 0, producer JLS-VII cell RNA;0, nonproducer RNA.

VOL. 29, 1979

1030 BESMER ET AL.

FIG. 7. Electrophoretic analyses of cytoplasmicRNA from VII-NP cells. A 5-tLg amount each ofpolyadenylic acid-selected cytoplasmic RNA fromVll-NP cells, Vll producer cells, and uninfectedJLS- V9 cells was electrophoretically separated on a

1% agarose methylmercuric hydroxide gel for 2.5 h at6 V/cm. The RNA in the gel was then transferred todiazobenzyloxymethyl paper, and the M-MuLV spe-cific bands were visualized by hybridization with 32P-

labeled M-MuLV cDNA. The exposure time for theautoradiography was 8 h. (a) Pure M-MuLV RNA;(b) VII-NP RNA; (c) Vl producer RNA; (d) JLS- V9RNA.

mic RNA extracted from Vll-NP was separatedby electrophoresis through denaturing methyl-mercuric hydroxide agarose gels. The RNA was

transferred from the agarose gel to diazoben-zyloxymethyl paper, an affinity paper for nucleicacids described by Alwine et al. (2). The virus-specific RNAs covalently linked to the paperwere then detected by hybridization with P-labeled M-MuLV cDNA. The largest viral RNAin the Vll-NP cells (Fig. 7, lane b) was 1,000nucleotides shorter than that of wild-type M-MuLV RNA (Fig. 7, lane a); the subgenomic 21SRNA was present in both the M-MuLV-produc-ing cells (Fig. 7, lane c) and the Vll-NP and wasapparently the same size in the two cell lines.Vll producer cells contained the regular ge-nomic 38S RNA, subgenomic 21S RNA, and an

RNA that comigrated with the larger virus-spe-cific RNA of the Vii-NP cells (Fig. 7, lane c).Uninfected cells showed no RNA bands by thisprocedure (Fig. 7, lane d).

The Vll-NP cells therefore appear to containa genome having a 1,000-nucleotide deletion.The deletion, though, does not affect the size ofthe 21S subgenomic RNA, implying that thedeletion occurs outside of the regions covered bythis RNA. The inability to detect the deletionby liquid hybridization experiments (Fig. 5 and6) is probably a consequence of its representingonly 11% of the 9,000-nucleotide genome. TheVll producer cells contained both full-length38S RNA as well as a species with most likelythe same deletion as the one found in the Vll-NP cells. Virus produced by the Vll cells alsocontains full-length 38S RNA and the deletedmolecule (P. Besmer, unpublished data).To determine whether the deleted genomic

RNA as well as the 21S subgenomic RNA wascontained in polyribosomes, the polyribosomefraction of the Vii and V11-NP cells was treatedwith or without EDTA and analyzed by sedi-mentation through sucrose gradients. Virus-spe-cific RNA in the "released messenger region" ofthe sucrose gradients (50 to 200S) was furtheranalyzed by sedimentation through sucrose-SDS gradients, and virus-specific RNA appear-ing in this region after EDTA disruption ofpolyribosomes was scored as functional mRNA.(15). By this criterion polyribosomes from theVll-NP and the Vii cells contained both ge-nome size 35 to 38S and subgenomic 21SmRNA's (Fig. 8). The deleted genome RNAfrom the Vll-NP cells is therefore found onpolyribosomes as well as the subgenomic 21SRNA.Can the virus genome in the V11-NP cells

be rescued? Previously we have shown thatuninfected V9 cells can be induced to producethe BALB/c endogenous xenotropic (MuLVx)and ecotropic (MuLVE) virus upon treatmentwith halogenated pyrimidines (8). The N-tropicMuLV does not form XC cell plaques whenassayed in an XC assay with NIH/3T3 cells asindicators (50). Vll-NP cells were treated withbromodeoxyuridine to determine whether thecompound could induce the endogenous V9 vi-ruses, which might then rescue or complementthe M-MuLV contained in the Vii-NP cells.The virus yield 2 days after removal of thehalogenated pyrimidine was determined (Table3). V9 cells upon induction produced 3 x 105focus-forming units of MuLVx per ml (as meas-ured by a focus assay on S+L- cat cells) and noXC cell plaques on NIH cells. The Vii-NP cellsupon induction produced 3 x 105 focus-formingunits of MuLVx per ml but also made 50 to 100PFU of virus per ml that gave XC cell plaqueson NIH cells. In an attempt to separate this lowamount of XC plaque-forming virus from the

J. VIROL.

.....

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MOUSE CELL LINE WITH DEFECTIVE M-MuLV GENOME 1031

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FIG. 8. Size of M-MuLV-specific mRNA in JLS-VI1 and Vll-NP cells. (a) JLS-Vll cells. Purifiedpolyribosomes were prepared from 1015-cm tissue culture dishes ofJLS-V1I cells at half confluency. Beforeextraction the cells had been treated with 1 pg of cycloheximide per ml for 30 min to increase the size of thepolyribosomes. The polyribosomes were divided in half, and one half was treated with 10 mm EDTA todisaggregate them. The two samples were thein sedimented in a sucrose gradient to display thepolyribosomes(not shown), and fractions containing the "rekased messenger" regions (50 to 200S) were pooled. RNA was

extracted from the two pooled samples and sedimented in a 10.5-ml 15 to 30% sucrose gradient in SDS buffercontaining 0.1% SDS. After centrifugation in a Beckman SW41 rotor for 12 h at 24,000 rpm and 25°C, thegradients were fractionated, and 100-pl portions of each fraction were concentrated by ethanol precipitationinto 50 IlI of 0.3 M NETES-0.1% SDS buffer. 3H-labeled M-MuLV cDNA (550 cpm) was added to eachconcentrated fraction and annealing was performed at 66°C for 18 h. After annealing, the samples were

digested with Sl nuclease, and the amount ofSl-resistant radioactivity was determined. The relative amountof virus-specific RNA present in each fraction was calculated from the percentage of cDNA hybridized as

before (a relative virus-specific RNA concentration of corresponds to 50% cDNA probe annealed) (13). Thedata from the two sucrose gradients are superimposed, and the locations of the 28S and 18S rRNA's(determined by monitoring the sucrose gradient for absorbance at 260 nm during fractionation) are alsoindicated. Symbols: 0, data from the EDTA treated polyribosomes; 0, data from the control polyribosomes.(b) Vll-NP cells. A similar experiment wasperformed with 1015-cm dishes of Vll-NP cells at half-confluency.

TABLE 3. Rescue ofM-MuLV from Vll-NP cellsupon induction with bromodeoxyuridinea

Cells XC plaques/ml MuLVx(FFU/ml)JLS-V9 0 3 x 105V11-NP 50-100 3 x 105

a Bromodeoxyuridine induction ofVl1-NP cells wasperformed as described previously (8). Two days afterthe removal of the bromodeoxyuridine-containing me-dium from the cultures, a 24-h harvest was collected,and the XC plaque titer on NIH/3T3 indicator cellsand MuLVx titer on cat S+L- cells were determined.FFU, Focus-forming units.

endogenous non-plaque-forming N-tropic virus,5 x 10i NIH cells were infected with the inducedvirus from NP cells. At 5 days after the infection,the supernatant was used to infect a new cultureof NIH cells, and this procedure was repeatedthree times. A virus stock was then preparedand titered on NIH and BALB/c cells. Twoindependent virus isolates derived were N-tropicand XC plaque-forming viruses and not NB-

TABLE 4. Characterization of virus isolated fromthe bromodeoxyuridine-induced virus of Vll-NP

cellsaVirus titer on:

Isolate NIH cells BALB/c 3T3 cells(XC plaques/ml) (XC plaques/ml)

1 2.6 x 105 6 x 1032 4.3 x 105 6 x 103

a NIH/3T3 cells were plated at 5 x 105 cells per 5-cm dish, and on the next day the cultures were infectedwith bromodeoxyuridine-induced virus from Vll-NPcells in the presence of 8 jg of polybrene per ml. At 5days after infection, a 24-h harvest was used to infecta new culture of NIH/3T3 cells, and this procedurewas repeated three times. From the third infectedculture, 5 days after the initial infection a 24-h harvestwas collected. The respective XC plaque titers oftheseisolates were then determined on NIH/3T3 cells andon BALB/c 3T3 cells.

tropic as would be expected forM-MuLV (Table4). These N-tropic viruses thus have the hostrange marker of the endogenous BALB/c N-

VOL. 29, 1979

1032 BESMER ET AL.

tropic virus (30, 40) and the large XC plaquemorphology marker ofM-MuLV (44). They maybe recombinants between the endogenous N-tropic MuLV and the defective M-MuLV in Vll-NP cells. Recombinants involving the Fv-1 hostrange marker and the XC plaque morphologymarker have been described previously (21).

DISCUSSIONOrigin of V11-NP cells. The V11-NP cell

line appears to have arisen from infection by a

deletion mutant of M-MuLV. This deletionprobably existed in the stock M-MuLV used toinfect JLS-V9 cells and generate the parentalJLS-Vll cell culture. Both the stable producerVll clone studied here and the V11-NP clonecontained this M-MuLV RNA lacking 1,000 nu-

cleotides; the producer also had full-size M-MuLV RNA. If Vll-NP had been derived as a

mutant from Vll, then the deleted RNA wouldprobably not have been found in the producercells.The Vll producer cells, once cloned, have

been stable producets for more than 100 gener-ations thereafter. Thus, there is no tendency tolose the nondeleted genome, and we concludethat the V11-NP cells probably did not ever

have a nondeleted genome. The Vll-NP cells

appear to have a slight growth advantage suchthat they could overgrow the producer cells inthe original mixed culture, and they are resistantto superinfection by M-MuLV. These two prop-erties most likely were essential for the isolationof the Vl1-NP cells.Nature and consequences of the deletion

in Vll-NP cells. The Vll-NP cells have bothdeleted genome-size RNA (vRNA) and normal-size 21S RNA on polyribosomes. They expressenv normally, making an intracellular Pr8O thatcleaves to a surface gp70. Thus, the 21S RNA,known to be the glycoprotein mRNA (54), ap-pears to be unchanged in structure and functionby the deletion. By contrast, no Pr659'9 or

Pr1809'"" is evident in the cells, implying thatthe deletion has interferred with their expres-sion. RNA indistinguishable from vRNA isknown to be themRNA for the gag-pol products(27, 33, 54). The P45 protein in the Vl1-NP cells

could be the product of the mRNA function ofthe deleted vRNA in the polyribosomes. If thisprotein is the N-terminal fragment ofthe nornalgag polypeptide, it should have p15, p12, andsome of p30. The deletion may therefore occurwithin the p30 gene. We are presently analyzingP45 further, but it is worth noting that thisfragment is just the size of the gag fragmentfound in the product of the Abelson MuLVgenome (56). In both Vl1-NP and Abelson

MuLV-infected cells, the product is immunopre-cipitated by anti-p30 antiserum but does notregister in radioimmunoassays (37). Thus, thedeletion mutant described here either could bethe parent of Abelson MuLV or could representa site at which deletions or substitutions fre-quently occur. The virus in Vll-NP cells mighteven have a partially substituted genome.An implication from this analysis is that the

splicing event that makes the 21S mRNA canoccur even though the intervening region hasbeen partly deleted. This conclusion is subject,however, to the reservation that different inte-grated viral genomes may give rise to the vRNAand to 21S mRNA in Vll-NP cells.Lack of VSV pseudotype production by

Vll-NP cells. Although Vll-NP cells havegp7O on their surface, numerous attempts toproduce VSV psuedotypes in these cells havefailed (Table 2). This result supports an earlierconjecture (55) that to have a glycoprotein in-corporated into the envelope of a virus requiresboth the glycoprotein and a submembrane pro-tein. The lack of the submembrane Pr65Vg inV11-NP cells presumably prevents gp7O fromparticipating in pseudotype formation.Mutability ofthe M-MuLV genome. In pre-

vious work (20, 35, 41, 42, 47), we and othershave characterized five nonconditional mutantclasses of MuLV's that are phenotypically dif-ferent from the deletion in V11-NP (see refer-ence 47). These other mutants arose from care-fully cloned MuLV's in one passage, implying avery high rate of spontaneous mutations duringMuLV passage. At least one of the five classesrepresented a deletion mutant. That deletion(M23) abolished both pol and env function butleftgag function intact (47). The ability to derivecell lines with a range of M-MuLV mutantsshould facilitate analysis of the roles of the var-ious gene products in the life of the virus. Thehigh rate of generation of mutants, however,must be considered in any explanations of thebehavior retrovirus-infected cells.

ACKNOWLEDGMENTSWe thank D. Arigoni, Eidgenossisch Technische Hoch-

schule, Zurich, Switzerland, for a generous gift of 1-[(m-nitro-benzyloxy)methyl]pyridium chloride, Poul Andersson for hishelp with the RNA blots, Richard Hynes for his advice withthe cell surface iodinations, and Daniel Clark for expert as-sistance.

This work was supported by Public Health Service grantsCA-15747 (H.F.), CA-14195 (core grant at the Salk Institute),and CA-14051 (core grant to S. Luria at M.I.T.) from theNational Cancer Institute and American Cancer Society grantno. VC-4I (D.B.). P.B. was supported by Public Health ServiceNational Research Service Award CA05083 from the NationalCancer Institute. D.B. is a Research Professor of the AmericanCancer Society.

J. VIROL.

MOUSE CELL LINE WITH DEFECTIVE M-MuLV GENOME 1033

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