Vol. 45, No. 3JOURNAL OF VIROLOGY, Mar. 1983, p. 995-10030022-538X/83/030995-09$02.00/0Copyright © 1983, American Society for Microbiology

Biological, Chemical, and Immunological Studies of RauscherEcotropic and Mink Cell Focus-Forming Viruses from JLS-V9

CellsALAN SCHULTZ,* ALAN REIN, LOUIS HENDERSON, AND STEPHEN OROSZLAN

Biological Carcinogenesis Program, National Cancer Institute-Frederick Cancer Research Facility,Frederick, Maryland 21701

Received 7 October 1982/Accepted 2 December 1982

Two murine leukemia viruses were isolated from JLS-V9 cells which had beeninfected with Rauscher plasma virus. One virus was XC positive and failed togrow on mink or cat cells and thus was an ecotropic virus. The other virus formedcytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. Theglycoproteins of the two viruses could be distinguished immunologically, bypeptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The MCF virus produced gp69, and the ecotropic virus producedgp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp7l) ofRauscher leukemia virus. Amino-terminal sequences of gp69 and gp7l weredetermined. The MCF sequence was distinct from the ecotropic sequence, butretained partial homology to it. The data show that the glycoproteins are encodedby related yet distinct genes. The protein structural data support the proposal thatMCF virus gp7O molecules have nonecotropic sequences at the amino terminus,with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCFvirus glycoprotein lacks a glycosylation site found at position 12 of the ecotropicsequence.

We have for some time been studying thebiosynthesis and post-translational modifica-tions of Rauscher murine leukemia virus (R-MuLV) gag and env gene products. The resultsfrom such studies in this and other laboratories(1, 2, 33, 34, 39) and primary sequence analysesof viral proteins (17, 18, 27, 41) make R-MuLVone of the best-characterized retroviruses fromthe point of view of protein chemistry. Onestructural problem, the presence of two envglycoproteins (gp69 and gp7l), has remainedunresolved since the first biochemical investiga-tions into R-MuLV (37). Whether the presenceof these two envelope glycoproteins reflectsheterogeneity in the modification and processingof the env gene product or represents anotherphenomenon has not been adequately ad-dressed.We have now analyzed the two proteins by

biochemical and immunological methods. Theresults of these studies indicated that they differin primary structure, and that the gp69 compo-nent carries antigenic determinants found onenvelope glycoproteins of mink cell focus-form-ing (MCF) viruses. A similar size differencebetween the ecotropic gp7O ofAKR virus and itsMCF virus derivatives has been described previ-ously (9). These results led us to look for an

MCF virus by biological cloning of viruses fromR-MuLV stocks grown in JLS-V9 cells. Thisanalysis resulted in the separation of an ecotro-pic and an MCF virus from R-MuLV stocksgrown in JLS-V9 cells.When MCF viruses were first discovered they

were described as a new class of retroviruswhich had dual ecotropic and xenotropic proper-ties (11, 14). Subsequently, many examples ofindependently arising MCF viruses have beendescribed, including one isolate from R-MuLV(38). Tryptic map comparison studies haveshown that MCF virus gp7O molecules containpeptides in common with both ecotropic andxenotropic viruses, which is consistent with theidea that MCF virus gp7O is a recombinantmolecule comprised of two domains (7). At thattime it was proposed that the amino-terminaldomain came from the ecotropic parent and thatthe carboxyl-terminal domain came from thepresumed xenotropic parent (6). However, anextensive series of DNA restriction maps hasrecently defined the extent of insertion of xeno-tropic-like sequences into the parental ecotropicgenome (4). From these maps the env gene ofnine MCF viruses appears to begin with a xeno-tropic virus-like sequence, and ecotropic se-quences occur only in the 3' end of gp7O or in

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p1SE. Similar conclusions have been reached bycomparison of oligonucleotide maps of viralRNA and env mRNA (8) and by peptide analysiswith monoclonal antibodies (28, 43).

Described here are the first results of primarystructure analyses of the proteins of an MCFvirus. The data demonstrate that the RauscherMCF (R-MCF) virus gp69 begins at its aminoterminus with a unique sequence, distinct fromthat of its ecotropic counterpart. A comparisonwith a deduced sequence from another MCFvirus (3) suggests that independent MCF virusisolates arose by substitution of very similarnonecotropic sequences.

MATERIALS AND METHODS

Cells and viruses. JLS-V9 mouse fibroblast cellsinfected with R-MuLV (44), CCL 64 mink lung cells(15), normal rat kidney cells (NRK), and SC-1 mousefibroblast cells (13) were obtained from the ViralResources Laboratory, Frederick Cancer ResearchFacility. Fischer rat embryo cells (FRE) infected withR-MuLV were obtained from Berge Hampar (NationalCancer Institute, Frederick Cancer Research Facility).NIH/3T3 cells were obtained from Donald Blair (Na-tional Cancer Institute), and PG-4 cat S+L- cells werea gift of D. Haapala (National Cancer Institute).Rauscher plasma virus, prepared from the spleens ofinfected mice, was obtained through the Office ofProgram Resources and Logistics, Division of CancerCause and Prevention (National Cancer Institute).

Radioisotopes and reagents. [35S]methionine (400 to900 Ci/mmol) and 2,5-diphenyloxazole were purchasedfrom Amersham Corp. (Arlington Heights, Ill.). V8protease came from Miles Laboratories Inc. (Elkhart,Ind.), and polyriboadenylate:oligodeoxythymidylate[poly(RA)-p(dT)12_18] came from PL Biochemicals(Kankakee, lll.). Protein A-Sepharose was purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). DEAE-dextran was obtained from Pharmacia Fine Chemicals(Piscataway, N.J.). Tunicamycin was obtained fromthe Lilly Research Laboratory (Indianapolis, Ind.)through the courtesy of Irving Johnson and RobertHamill.

Antisera. Antiserum to purified gp70 of R-MuLVwas prepared in goats as previously described (41).Goat antiserum to purified gp7O of Friend MCF virus,absorbed with Friend ecotropic virus, was generouslyprovided by Sandra Ruscetti (National Cancer Insti-tute).An antiserum to FRE cells infected with R-MuLV

was prepared in Fischer rats in the following way.Rinsed R-MuLV-infected FRE monolayers werescraped into Dulbecco phosphate-buffered saline(PBS) and pelleted. The cell pellet was suspended inan equal volume of PBS and divided in half. One half(whole cells) was stored frozen. The other half (lysedcells) underwent six freeze-thaw cycles and brief soni-cation. Rats were immunized simultaneously withwhole cells and lysed cells as follows. Whole cellswere inoculated intraperitoneally, and lysed cells inFreund complete adjuvant were injected subcutane-ously in several sites. Animals were boosted twice byboth inoculation routes at weekly intervals and were

sacrificed by heart puncture 2 weeks after the finalboost.

Cell culture and virology. Calf serum and fetal calfserum were purchased from GIBCO Laboratories(Grand Island, N.Y.). Minimal essential medium, Dul-becco reinforced essential medium, and methionine-free minimal essential medium were obtained fromFlow Laboratories (Rockville, Md.).

Cells were grown on Corning plastic ware in medi-um supplemented with 10% heat-inactivated calf orfetal calf serum, penicillin (100 IU/ml), and streptomy-cin (100 ,ug/ml) under an atmosphere of 5% CO2 in air.Cultures were regularly tested by Richard DelGiudice(National Cancer Institute, Frederick Cancer Re-search Facility) for mycoplasma contamination andwere maintained mycoplasma free.

General tissue culture and virological proceduresand virus-cloning procedures have been described(30). Infectivity assays for the nonecotropic MuLVwere performed with the PG-4 line of cat S+L- cells(D. Haapala, manuscript in preparation).

Radiolabeling of cell cultures. Monolayers were la-beled with [35S]methionine in methionine-free minimalessential medium as described previously (34). Tunica-mycin, when included, was present at 5 ,ug/ml for 1 hof pretreatment, but not during the radiolabeling peri-od itself.To prepare radioactive proteins for subsequent puri-

fication and analysis, cells were labeled in suspension.A T150 flask was preincubated as a monolayer inamino acid-free medium for 15 min and then rinsedwith warm PBS. Trypsin-EDTA (GIBCO) was added,and flasks were incubated at room temperature untilcells were loosened. Trypsinization was terminatedwith PBS containing 10% dialyzed calf serum, and thecells from one T150 flask were transferred with 15 mlof PBS containing 10% dialyzed calf serum to a conicalcentrifuge tube. After centrifugation at 800 x g, thepellet was suspended in 2 ml of amino acid-freemedium containing 3H-amino acids (100 ,uCi/ml) andincubated at 37°C for 0.5 to 2 h. The suspended cellswere washed by centrifugation three times with coldPBS and dissolved with lysing buffer as describedbelow.

Immunoprecipitation and analysis of radioactive pro-teins. At the end of a labeling period, cells were lysedwith a buffer containing Nonidet P-40 and deoxycho-late as previously described (35). Recovery ofimmunoprecipitates with protein A-Sepharose and so-dium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) gradient slab gels by the method ofLaemmli (21) has been described previously (33).Radioactive bands were located by scintillation auto-radiography (22).

Peptide mapping. One-dimensional peptide mapswere performed on 7.5 to 17% gradient SDS-polyacryl-amide Laemmli gels after digestion of protein with V8protease in 0.1 M NH4HCO3 (pH 7.8) for 16 h at 37°C.

Purification of viral proteins by rpHPLC. All high-pressure liquid chromatography (HPLC) was per-formed with a Waters Associates (Milford, Mass.)liquid chromatograph equipped with two model 6000M solvent delivery pumps, a model 660 solvent pro-grammer, a model 450 variable wavelength detector,and a reversed-phase ,. Bondapak phenylalkyl columnobtained from Waters Associates. Solvents for re-versed-phase HPLC (rpHPLC) were 0.1% trifluoro-

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acetic acid in quartz-distilled water as the aqueousphase and acetonitrile with 0.1% trifluoroacetic acid asthe organic phase as previously described (19).Amino acid composition and protein sequencing.

Samples for amino acid analysis were hydrolyzed invacuo with 0.2 ml of 6 N HCI containing 0.1% liquidphenol at 110°C for 24 h. Analysis was performed on aDurrum D500 amino acid analyzer equipped with aPDP 8/A computing integrator.Semiautomated microsequence analysis with Ed-

man degradation was performed on intact proteinswith a Beckman sequencer model 890C as describedelsewhere (5). Phenylthiohydantoin amino acids wereidentified and quantitated by HPLC (16).

RESULTS

Structural comparison of env glycoproteins ofR-MuLV. Our first firm clue that Rauscher gp69and gp7l did not result from variation in theextent of carbohydrate processing amonggPr85env forms came in studies with tunicamy-cin, an inhibitor of glycosylation. In Fig. 1 isshown the effect on gPr85env synthesis of varioustunicamycin concentrations from 0 (lane 1) to 5(lane 4) ,ug/ml. The unglycosylated forms of theprecursor polyprotein, Pr68env, which were pro-duced retained the size difference characteristicof the glycosylated forms. The apparent molecu-

UI, Uw gPr85

o t

1 2

Pr68

4

FIG. 1. Doublet env gene-encoded proteins fromR-MuLV grown in JLS-V9 cells. The proteins weredetected in the presence of tunicamycin. Incorpo-ration of [355]methionine (5 ,uCi/ml, 15 min) wasanalyzed by SDS-PAGE after immunoprecipitationwith anti-R-MuLV gp7O serum. Lanes: 1, control; 2, 1,ug of tunicamycin per ml; 3, 3 ,ug of tunicamycin perml; 4, 5 ,ug of tunicamycin per ml.

U4'-

-W -921-

_ -691'

mm -4'6?1

- 29K

-1 2;.5-

FIG. 2. One-dimensional map of env precursorpolyproteins digested with V8 protease. JLS-V9 cellslabeled in suspension with a 3H-amino acid mixture(100 .Ci/ml) for 2 h were immunoprecipitated withanti-gp7O serum. The two env precursor polyproteinswere each recovered from the immune precipitateafter SDS-PAGE by slicing from the gel and digestedwith V8 protease as described in the text. Analyzed ona 7.5 to 17% gradient SDS-PAGE gel is the largerprotein gPr85'v (lane 1) and the smaller proteingPr83 nv (lane 2). Size markers are phosphorylase a(92,000 daltons), bovine serum albumin (69,000 dal-tons), ovalbumin (46,000 daltons), carbonic anhydrase(29,000 daltons), and cytochrome c (12,300 daltons).

lar weight difference was about 2,500 daltons.Similar results with 2-deoxyglucose to inhibitglycosylation have previously been obtained inFriend (F-MuLV) (25). Apparently differencesin the carbohydrate structure are not responsiblefor the size difference.

This hypothesis was confirmed by one-dimen-sional peptide mapping studies of proteins isolat-ed from JLS-V9 cells. Limited digestion with V8protease indicated that the two glycoproteinshad nonidentical peptide chains. In this case,gPr85enV molecules labeled with tritiated aminoacids were cut from preparative SDS-PAGEgels, digested, and rerun. The major peptidesvisualized in the autoradiogram from the largergPr85env in Fig. 2 (lane 1) are quite different insize from those of the smaller gPr83e"V (lane 2).

Purification and amino acid composition ofgp69 and gp7l. gp69 and gp7l glycoproteinsdiffered sufficiently in physical properties to beresolved by rpHPLC. Figure 3 shows the sepa-ration of viral proteins eluting from a IL Bonda-pak phenylalkyl column in an increasing gradi-ent of acetonitrile in 0.1% trifluoracetic acid.Proteins elute in the order of increasing hydro-phobicity (plO, p12, gp69, gp7l, p30, p15). Pro-teins occurring in each peak have been identifiedby SDS-PAGE, immunological tests, and aminoacid sequencing (data not shown). The pl5E canbe eluted under more stringent conditions with

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FIG. 3. Elution profile of R-MuLV proteins sepa-rated by rpHPLC. The earliest fractions to be elutedare shown at the right side of the figure, and theacetonitrile concentration at each point in the complexgradient used to elute the proteins is indicated by thetriphasic solid line extending across the profile. Pro-teins occurring in the peaks have been identifiedpreviously (20).

propanol (data not shown) as described previ-ously (20).

In the plO region there are three peaks. How-ever, analysis shows that only the major peak isplO; the identification of the other two peaks willbe reported separately (Henderson et al., manu-script in preparation). Also, the p12 resolvesinto two peaks; the reason for this will bedescribed below.Amino acid compositions of gp69 and gp7l

proteins purified by rpHPLC are given in Table1. The gp69 differs from gp7l most obviously inthe tyrosine, methionine, and histidine content.Two comments can be made concerning the

TABLE 1. Amino acid compositions of R-MuLVgp69 and gp71l

Amino acid gp69 gp7l

Asp 36 38Thr 35 40Ser 23 28Glu 36 31Gly 39 39Ala 26 27Val 26 27Met 5 2Ile 11 9Leu 32 40Tyr 13 21Phe 9 6His 7 14Lys 19 18Arg 20 18

a Data are presented as moles per mole, rounded tothe nearest integer.

small amount of gp69 relative to gp7l in Fig. 3when from Fig. 1 the precursor proteins appearto be synthesized in roughly equal amounts.First, from the amino acid composition (Table 1)it is seen that the gp69 contains 2.5 times asmuch methionine as gp7l, so that its relativeamount will be overestimated in an [35S]me-thionine labeling. Second, gp7l may be incorpo-rated into virions more efficiently than gp69, orgp69 may attach less firmly to the particle; thiscould explain the phenomenon of "genomicmasking" (10, 12).Immunological characterization of R-MuLV

env glycoproteins. Ruscetti et al. (32) have de-scribed the preparation of an MCF virus-specificantiserum. When this serum was reacted with R-MuLV particles, it was found to precipitate thegp69, but not the gp7l, env protein (Fig. 4, lane3). In contrast, a Fisher rat serum producedagainst R-MuLV-infected cells as describedabove precipitated gp7l, but not gp69 (Fig. 4,lane 2). Small amounts of the gag proteins Pr65and p30 were also precipitated by this serum.Other tests (data not shown) with this antiserumshowed its anti-env antibody to be primarilygroup specific (F-MuLV, Moloney MuLV, or R-MuLV).

ggp71gp69

-p30

1 2 3FIG. 4. Immunoprecipitation analysis with three

different antisera of R-MuLV grown in JLS-V9 cells.Cells were labeled with [35S]methionine (10 ,uCi/ml)for 30 min and chased overnight. Virus, recoveredfrom the medium by pelleting through a 20o sucrosecushion, was Isyed in lysing buffer containing 0.1%SDS and immunoprecipitated with one of three anti-sera. Lanes: 1, goat antiR-MuLV gp7O serum; 2, ratanti-R-MuLV serum prepared as described in the text;3, goat anti-F-MCF virus gp7O serum absorbed withecotropic F-MuLV.

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Taken together, these results complement thestructural comparisons discussed above, sincethey demonstrate that each of the two envproteins in R-MuLV particles possesses antigen-ic determinants not present in the other compo-nent. They also raise the possibiijty that the gp69is an MCF virus-specific env protein.

Isolation and characterization of two virusesfrom R-MuLV. The structural and antigenic dif-ferences between gp69 and gp7l, as well asprevious work by Murray and Kabat (25) on F-MuLV, were all consistent with the hypothesisthat these two proteins are encoded by distinctviral genomes. We therefore separated thesegenomes biologically. One component was iso-lated by passage at low multiplicity of infectionof the JLS-V9 R-MuLV stock in NRK rat cells;this virus was found to be an ecotropic MuLV,since it gave the same titer in the XC test as inthe mouse S+L- focus assay and formed no foci

gPr85_, 4.1 -92KgPr83 - . _gpll -

gp69" 1 0 -69K

q -46K

_ -29K

-12K

1 2 3 4 5 6FIG. 5. Immunoprecipitation analysis of R-MuLV

grown in JLS-V9 cells and of cloned R-MCF virus andecotropic R-MuLV with goat anti-R-MuLV gp70 se-rum. Incorporation of [35S]methionine (5 ,Ci/ml) pro-ceeded for 15 min in each of the three cell lines. Lanes1, 3, and 5 represent immunoprecipitations performedat the end of the labeling period. Lanes 2, 4, and 6represent immunoprecipitations performed on identi-cal flasks which had been chased for 2 h. Lanes: 1 and2, R-MuLV grown in JLS-V9 cells; 3 and 4, ecotropicR-MuLV grown in NRK cells; 5 and 6, R-MCF virusgrown in NIH 3T3 cells. Size markers are as describedin the legend to Fig. 2.

TABLE 2. NH2 terminal sequence analysis ofR-MCF gp69

Cycle Amino acid Yielda Cycle Amino acid Yield

1 Val 1.26 14 Trp 0.262 Gln 1.34 15 Arg 0.413 His 0.51 16 Val 0.484 Asp 0.79 17 Thr 0.105 Ser 0.19 18 Asn 0.376 Pro 0.41 19 Leu 0.307 His 0.38 20 Met 0.448 Gln 0.66 21 Thr 0.169 Val 0.73 22 No assignment10 Phe 0.96 23 Gln 0.3311 Tyr 0.28 24 Thr 0.1112 Val 0.72 25 Ala 0.3713 Thr 0.18a Yield of PTH-amino acid, expressed in nanomoles

and corrected for background.

on cat S+L- cells. Although this virus was notcloned by endpoint dilution, an infectious centerassay, performed in parallel with the proteinanalysis described below, showed that <0.1% ofthe infectious particles produced by these cellsformed foci on cat S+L- cells.A second component was isolated from the

JLS-V9 R-MuLV stock by first passaging thevirus on CCL64 mink cells to enrich for noneco-tropic MuLV and then infecting 3T3FL cellswith a limiting dilution of the mink-grown vir-us.This component was an MCF virus as shownby its interference properties (see Tables 1 and 2in reference 29); by the fact that it gives equiva-lent titers on mouse and cat S+L- cells, but doesnot form XC plaques; and by formation of foci(data not shown) in the mink cytopathic focusassay (14). This MCF virus may be similar oridentical to the isolate described by van Griens-ven and Vogt (38).

Figure 5 shows a characterization of the envglycoproteins produced by the two viruses,compared to those of the R-MuLV complex.The ecotropic MuLV produces a single gPr85envprecursor (Fig. 5, lane 3) which can be chasedinto gp7l (lane 4), whereas the MCF virusproduces a gPr83YPV which is converted intogp69 (lanes 5 and 6). The gag polyproteinsPr65gag and gPr80gaB of the two viruses wereindistinguishable by SDS-PAGE (data notshown).

Analysis of R-MCF virus proteins. Proteins forstructural analysis were isolated by rpHPLC asin Fig. 4. An amino-terminal sequence analysisof gp69 has been completed through 25 cycles.The amino acids assigned at each cycle, andtheir recovery, are summarized in Table 2. Theresulting sequence is aligned with the previouslypublished result for R-MuLV gp7l in Fig. 6.Sequences in common are boxed, and a glycosy-

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gp69 vYt-Hn_*- S-P-JH--V-eF-Y-VjiT-W R. V-T-N L-M-T-x O-T-A

gp71 A-A-P-G-S- S-P-H-04-VY-N-1. T-W O. V-T-N -G-*_n-R1 0-T-A.

5 10 15 20 25

FIG. 6. Sequence comparison of R-MuLV gp69and gp7l. The sequence is given in the following one-letter code: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H,His; I, Ile; K, Lys; L, Leu; M, Met; N, Asp; P, Pro; Q,Gln; R, Arg; S, Ser; T, Thr; W, Trp; X, unidentifiedresidue; Y, Tyr. (*) designates a gap introduced tomaximize homology. The symbol CHO indicates gly-cosylation confirmed by carbohydrate analysis (un-published data).

lation site confirmed in gp7l (unpublished data)at position 12 is indicated. In Fig. 7 the R-MCFvirus gp69 is aligned with an F-MuLV gp69sequence (24) and a recently obtained glycopro-tein sequence for Moloney MCF (M-MCF) virusdeduced from nucleotide sequence of the env

gene region (3).The plO of R-MCF virus has an amino acid

composition identical to that for ecotropic R-MuLV (data not shown). Preliminary analysishas also not shown any difference in the p15 andp30 proteins. However, an amino-terminal se-quence analysis of R-MCF virus p12 (data notshown) providing the sequence of the first 50residues shows that it is virtually identical tothat previously determined for R-MuLV, exceptfor the single substitution of isoleucine for valineat position 24, where microheterogeneity hadbeen detected (40). Thus the two peaks in thep12 region of Fig. 4 represent separation ofecotropic and MCF virus p12 by rpHPLC.Those two peaks differed in amino acid composi-tion; specifically, the minor peak contained iso-leucine, whereas the major peak did not (datanot shown).

Analysis of Rauscher plasma virus. The JLS-V9 cells were infected with Rauscher plasmavirus in 1965 (44). The possibility that the viruswe are calling R-MCF virus might have arisen inthe JLS-V9 cells and not come from the original

F-MuLV gp69 V-A-P-x-T-P-P-0-V-F-Y-V-T-W-R-V-x-N-L-M-

R-MCF gp69 V-(0-H-n-S-P-H-0-V-F-Y-V-T-W-R-V-T-N-L-M-T-x-Q-T-A-

M-MCF gp7n V-n-H-n-S-P-H-0-V-F-N-V-T-W-R-V-T-N-L-M-T-G-0-T-A-

5 1n 15 20 25

FIG. 7. Sequence comparison between R-MCF vi-rus gp69, M-MCF virus gp7O, and F-MuLV gp69. Theone-letter code used is as described in the legend toFig. 6. Note that the gap introduced at position 5 ofgp69 in Fig. 6 to maintain homology with gp7l is notneeded for the alignment of MCF virus glycoproteinsand has been eliminated here. The F-MuLV gp69sequence is from Linder et al. (24); the M-MCF virusgp70 sequence is from Bosselman et al. (3).

Rauscher stocks was investigated by looking forgp69 in Rauscher plasma virus. To do this,plasma virus was used to infect SC-1, a murinecell line in which all known murine retroviruseswill grow. Successful infection, as judged byreverse transcriptase activity, was obtained af-ter two passages. The presence of gp69 wasexamined by [ S]methionine incorporation andimmunoprecipitation with goat anti-R-MuLVgp7O. The results (Fig. 8) show that gp69 andgp71 can both be detected after infection withRauscher plasma virus.

DISCUSSIONOur studies show that in JLS-V9 cells infected

with R-MuLV, the two envelope glycoproteinswhich differ in size (gp 69 and gp7l) are synthe-sized by an ecotropic and MCF pair of viruses.The virus producing gp71 is ecotropic because itfails to infect cat or mink cells and gives equiva-lent titers on XC cells and in the S+L- assay.The other virus, producing gp69, is assigned tothe MCF virus class not only because it formscytopathic foci on mink cells but because itinterferes with and is interfered with by onlyMCF viruses in an infectivity assay (29). Eachvirus produces only a single envelope glycopro-

gPr85gPrBO-

Pr65-

-92K

-69K.IIg4-.

-46K

-29K

1 2 3 4 5 6

FIG. 8. Precursor polyproteins encoded by gagand env genes detected after infection of SC-1 cellswith Rauscher plasma virus. Rauscher plasma virusgrown in SC-1 cells (lanes 1, 2, and 3) or R-MuLVgrown in JLS-V9 cells incorporated [355]methionine (5,uCi/ml) for 15 min. Three sera were used for immuno-precipitation; anti-p30 antiserum (lanes 1 and 4), anti-R-MuLV gp7O serum (lanes 2 and 5), and monoclonalanti-p15(E) serum (lanes 3 and 6).

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tein and precursor polyprotein; no size heteroge-neity is detectable by SDS-PAGE (Fig. 5).Other examples of heterogeneity in glycopro-

teins of ecotropic and MCF pairs of viruses havebeen reported. For example, a genetic origin forgp69 and gp7l of F-MuLV growing in Evelinecells (25) was proposed. In that case, cloning thevirus in murine cells gave rise to two kinds ofclones. One clone produced only gp7l, andothers produced both gp69 and gp7l. The virus-es were not further characterized biologically.MCF viruses arising during spontaneous leuke-mogenesis in AKR mice have also been recog-nized to have glycoproteins smaller than theirecotropic counterparts (9). However, this is nottrue of all ecotropic and MCF virus pairs, sincethe glycoproteins ofM-MCF virus and ecotropicM-MuLV are identical in size by SDS-PAGEand comigrate with R-MCF virus gp69 (data notshown).MCF viruses are derived by substitution of

nonecotropic sequences for ecotropic env genesequences. Previous studies have placed thenonecotropic moiety at either the carboxyl-ter-minal (26) or the amino-terminal (3, 4, 8, 28) endof gp7O. Our sequence data (Fig. 6) show thatthe amino-terminal sequences of R-MCF virusgp69 and ecotropic R-MuLV gp7l share 13 iden-tities in the first 25 residues; thus, the twoproteins are related, but not identical in thisregion. This homology shows that the two se-quences represent equivalent regions of mole-cules encoded by the same gene family; howev-er, the difference between them suggests that thesequences in the amino-terminal portion of R-MCF virus gp69 are the nonecotropic se-quences. This conclusion is reinforced by com-parison with the much higher homology foundwithin the ecotropic MuLV family: F-MuLV,Moloney MuLV, R-MuLV, and Akv env pro-teins share 21 identities in the first 25 residues(4a, 17, 20a, 23, 36). In addition, the virtualidentity ofR-MCF virus and ecotropic R-MuLVp12 sequences (49 of the first 50 residues) showshow similar the two viruses are outside the envregion shown in Fig. 6. This observation isparticularly significant in light of the variationfound in p12 sequences of ecotropic viruses: R-MuLV and M-MuLV p12 differ by 13 of the first50 residues (40). When considered together,these comparisons provide strong evidence thatthe nonecotropic sequences of R-MCF virusinclude the amino-terminal region of gp69,whereas the single deviation found in p12 ispresumably due to a point mutation which oc-curred since the substitution event that gave riseto R-MCF virus. From data available in thisstudy, it is not possible to determine the locationof the carboxy-terminal end of the substitutionin the R-MCF virus genome.

As has been noted (29) the substitution ofnonecotropic for ecotropic env sequences prob-ably confers a strong selective advantage upon aviral genome in the viremic animal, since itenables the virus to enter cells via an alternativereceptor. The fact that MCF and ecotropic vi-ruses utilize different receptors in fibroblastsalso means that neither virus affects the spreador maintenance of the other in tissue culture.Thus, we presume that the two viruses stablycoexisted in biologically active form in JLS-V9cells since the culture was infected withRauscher plasma virus in 1965 (44). It seemslikely that R-MCF virus, as well as ecotropic R-MuLV, was present in Rauscher plasma virus,since both gp69 and gp7l were easily detectedshortly after passage of Rauscher plasma virusin SC-1 cells (Fig. 8), and R-MCF virus has beenisolated directly from plasma virus (38).Although all known MCF viruses interact with

the same receptor on NIH/3T3 fibroblasts (29),they vary widely in their effects in vivo; theexplanation for this biological diversity is notknown. It was therefore of interest to comparethe R-MCF virus gp69 sequence with otherknown MCF virus env protein sequences, todetermine the degree of similarity of differentMCF virus isolates. Figure 7 shows the R-MCFvirus gp69 sequence and a very recently de-duced gp70 sequence for M-MCF virus (3).(Experimentally, M-MCF virus gp70 comigratesin SDS-PAGE with R-MCF virus gp69; thedesignation gp7O is taken from the reference andis not intended to denote the size of the mole-cule.) Alignment of the M-MCF virus deducedsequence with that obtained from the R-MCFvirus glycoprotein predicts that M-MCF virusgp7O will also have an amino-terminal valineresidue and shows that the R-MCF and M-MCFvirus sequences are highly homologous (23 iden-tities out of 25 residues). Also included in Fig. 7is the sequence of gp69 isolated from an F-MuLV gp69-gp7l mixture (24). From its homol-ogy to R-MCF and M-MCf, viruses, it is nowreasonable to assume that it represents an MCFvirus glycoprotein also. Preliminary sequencingstudies of env genes of AKR MCF247 (C. Hol-land, personal communication) and ofthe Friendspleen focus-forming virus (L. Wolff, personalcommunication) indicate that these viruses areas homologous to R-MCF virus in this region asM-MCF virus is. It thus appears that thesedifferent viruses have acquired nearly identicalgene substitutions in the amino-terminal regionof gp70, despite the fact that they arose indifferent mouse strains by recombination withdifferent ecotropic MuLVs.The near identity in the amino-terminal gp7O

sequences of different MCF viruses suggeststhat these substituted env sequences comprise a

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1002 SCHULTZ ET AL.

family of homologous genes analogous to thefamily of murine ecotropic env genes. Thisraises the possibility that differences in the bio-logical effects of these viruses, insofar as gp7Ostructure determines their pathogenicity, will bedue to variations in the extent of substitution orsubtle conformational or limited sequence vari-ances. The M-MCF virus gp7O sequence has anasn-val-thr sequence at positions 11 through 13(Fig. 7), a presumed glycosylation site. Thisglycosylation site is found in all ecotropic virus-es for which sequence is known (4a, 17, 20a, 23,24, 36), and analysis of ecotropic R-MuLV gp7lhas shown that the site actually is glycosylated(unpublished data). In contrast, R-MCF virusgp69 and gp69 of the F-MuLV complex (24) havetyrosine substituted for asparagine at that posi-tion (Fig. 7), thus eliminating that glycosylationsite. An oligosaccharide chain would be expect-ed to have a strong influence on the conforma-tion of the gp7O molecule, and this differencebetween M-MCF and R-MCF viruses, which areotherwise highly homologous in this region, issurprising. It is intriguing to note that R-MCFand F-MCF viruses cause erythroleukemia (31,38), whereas M-MCF virus is associated with T-cell leukemia (42). One possibility is that theMCF virus glycoproteins in some way confertarget organ specificity on the viruses and that,although fibroblasts do not discriminate betweenR-MCF and M-MCF viruses, hematopoieticcells may. The determination of the completeamino acid sequences for all these and otherMCF virus glycoproteins is needed to relatemore accurately the differences in primary struc-ture to the variations in the pathological mani-festation of these highly related leukomogenicagents.

ACKNOWLEDGMENTS

We thank R. A. Bosselman, C. Van Beveren, and I. Vermafor permitting us to see the M-MCF virus env gene sequencebefore publication, and we thank W. Koch, G. Hunsmann,and R. Friedrich for permitting us to see the F-MuLV env genesequence before publication. We also thank Gary Smythers,Sally Lockhart, Anne Soria, Melody McClure, and DorothyCavanaugh for excellent technical assistance.

This research was sponsored by the National Cancer Insti-tute, under Public Health Service contract NO1-CO-23909with Litton Bionetics, Inc.

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