JOURNAL OF VIROLOGY, Sept. 1977, p. 700-707Copyright © 1977 American Society for Microbiology

Vol. 23, No. 3Printed in U.S.A.

Evidence for Recombination Between N- and B-TropicMurine Leukemia Viruses: Analysis ofThree Virion Proteins

by Sodium Dodecyl Sulfate-PolyacrylamideGel Electrophoresis

JOHN SCHINDLER, RICHARD HYNES, AND NANCY HOPKINS*Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication 29 March 1977

We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis toanalyze the virion proteins of an N- and a B-tropic C-type virus derived from theBALB/c mouse and 21 putative recombinants, designated XLP-N viruses, ob-tained from seven crosses between these N- and B-tropic viruses. All the XLP-Nviruses are N-tropic but possess the XC plaque morphology of their B-tropicvirus parent. Three virion proteins, p15, p30, and gp7O, of the parental viruseseach differ in electrophoretic mobility. Two recombinants were found thatpossess a p15 that comigrates with p15 of the B virus; 19 possess a p15 thatcomigrates with N virus p15. Sixteen recombinants possess a gp7O that migrateslike the gp70 of the B virus; four have gp70 with an electrophoretic mobility likethat of the N virus gp7O. All 21 recombinants possess a p30 that comigrates withp30 of their N virus parent. Given the origin and phenotype of XLP-N viruses,these results would seem to provide good evidence that these viruses are recom-binants.

Naturally occurring ecotropic murine leuke-mia viruses can be classified according to theirhost range as either N-tropic or B-tropic de-pending upon whether they grow preferentiallyon cells derived from NIH Swiss mice (N-type)or BALB/c mice (B-type), respectively (8).Inbred strains ofmice can be classified as eitherN- or B-type, and this phenotype is determinedby a gene called Fv-1 (22, 23; reviewed in refer-ence 19). Restriction toward a virus of the"wrong" tropism is inherited as a dominanttrait. The restriction of viral growth on a cell ofincompatible Fv-1 type is relative rather thanabsolute and occurs after virus absorption andpenetration but before integration of proviralDNA (13, 16, 17, 27). The viral determinants ofN- or B-tropism have not yet been identified(11, 24). The Fv-1 gene plays a role in determin-ing susceptibility to leukemogenesis by N- or B-tropic murine leukemia viruses presumably byretarding the spread and resulting viremia of avirus of the wrong tropism (see 19).Using plaque morphology (9, 25) and N- and

B-tropism as genetic markers, Hopkins et al.(12) have presented biological evidence for re-combination between an N-tropic virus thatmakes small XC-plaques (SP-N) and a B-tropicvirus that makes large plaques (LP-B), bothderived from the BALB/c mouse: after coinfec-tion with SP-N and LP-B they obtained viruses,called XLP-N, that are N-tropic but make large

XC plaques. Hopkins et al. (10) have also ob-tained evidence based on antigenicity, thatXLP-N viruses are recombinants. We were in-terested in extending this biochemical analysis,since analysis of a number of recombinantsmight prove useful in identifying and mappingthe viral determinants of XC plaque morphol-ogy and N- and B-tropism. Famulari,O'Donnell, and Fleissner (personal communica-tion; see 11) have shown that three virion pro-teins of some N- and B-tropic virus isolatesderived from BALB/c mice can be distinguishedby their mobility on sodium dodecyl sulfate(SDS)-polyacrylamide gels. These are p15, aninternal virion protein, p30, the major struc-tural protein of the capsid of the core shell, andgp7O, the major envelope glycoprotein which isresponsible for specific absorption of C-type vi-ruses to cells. O'Donnell and Stockert (20) haveshown that these viruses also differ in theirability to induce G1Q, an antigenic determinantbelieved to reside on gp7O (28). These observa-tions provided us with a means of asking if theputative recombinants obtained by Hopkins etal. (12) possessed p15, p30, and gp7O proteinswith the electrophoretic mobility characteristicof their N- or B-tropic virus parent. Here wepresent the results of SDS-polyacrylamide gelelectrophoresis analysis of virion proteins of 21XLP-N viruses obtained from seven independ-ent crosses between SP-N and LP-B viruses.

700

VIRION PROTEINS OF N- AND B-TROPIC VIRUSES

Analysis of these viruses for the ability to in-duce the Gx antigen has been reported previ-ously (10).

MATERIALS AND METHODSCells, viruses, and media. Sc-1 cells (7) were

grown in Dulbecco modified Eagle medium with10% inactivated fetal calfserum and antibiotics. Theorigin of SP-N (9), LP-B (B-clone-18) (15), and XLP-N viruses (12) has been described. XLP-N viruseswere purified either by plaque purification (12) or bythe microtiter method (26).

Virus purification and gel electrophoresis. Thepreparation of virion proteins for gel electrophoresishas been described (11). Briefly, virus was purifiedby banding in sucrose gradients and then pelleted.Electrophoresis of virion proteins was on Laemmli(18) slab gels as described (11). Immunoprecipitationof the p15 virion protein was performed with goatanti-AKR p15 serum using Nonidet P-40-solubilizedvirus followed by precipitation with pig anti-goatimmunoglobulin G. The antibodies were a gift fromNancy Famulari.

RESULTSParental viruses SP-N and LP-B possess

p15's, p30's, and gp7O's that each differ inelectrophoretic mobility on SDS-polyacryl-amide gels. Virus was purified from culturefluids of Sc-1 cells (a cell line that does notexhibit Fv-1 gene restriction) infected witheither SP-N or LP-B virus and disrupted withSDS, and the proteins were separated by gelelectrophoresis. Figure 1 shows a slab gel onwhich the virion proteins of SP-N and LP-B arevisualized by Coomassie brilliant blue staining.The identification of p30 is based on publisheddata (1) and on a comparison with molecularweight markers (Fig. 1, track 1). The identifica-tion of the p15 band is based on unpublishedobservations of N. Famulari and E. Fleissner(personal communication), on published data(1, 14), on a comparison with molecular weightmarkers, and on the fact that this band can beprecipitated using antiserum specific for p15(unpublished data). It can be seen in Fig. 1 thatthe p15 and p30 proteins of SP-N and LP-B eachdiffer in electrophoretic mobility: the p30 of SP-N runs slightly ahead of p30 of LP-B whereasthe N virus p15 migrates slightly behind thep15 of LP-B. These differences were observednot only on the standard Laemmli gel system(18), but also on phosphate-buffered SDS gels(6), 6 M urea phosphate-buffered SDS gels, 6 Murea Laemmli gels, and low-bis Laemmli gels(5) (unpublished data).gp7O was visualized by autoradiography of

gels containing virion proteins labeled with 14C-amino acids (Fig. 2). The gp70 was identified bycomparison with molecular weight markersand by the fact that this band could be labeled

-p30

A:,;.:: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......

1 2 3 4 5FIG. 1. Comparison of p30 and p15 virion pro-

teins ofparental viruses SP-N and LP-B. Figure 1 isan 8 to 24% slab gel stained with Coomassie brilliantblue. Samples are as follows: 1, Marker mix: phos-phorylase A, 90,000 daltons; catalase, 62,000 dal-tons; creatine kinase, 40,000 daltons; carbonic anhy-drase, 29,000 daltons; cytochrome c, 12,700 daltons;2 and 4, SP-N; 3 and 5, LP-B.

with [14C]glucosamine (unpublished data). Thegp7O of SP-N migrates ahead of the gp7O of LP-B. The differences in electrophoretic mobilitybetween the p30's and the p15's of SP-N and LP-B can also be seen on the autoradiogram in Fig.2, but the difference is not as clear as in Fig. 1since the exposure time necessary to detectgp70 results in overexposure of the p15 and p30bands.Virion proteins of recombinants between

SP-N and LP-B. Having determined that thepi5, p30, and gp70 proteins of SP-N and LP-B,the parental viruses used to generate the puta-tive recombinants, could be distinguished as

701VOL. 23, 1977

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702 SCHINDLER, HYNES, AND HOPKINS

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ing p30 bands. This gel shows the p30's of sixrecombinants, and, for comparison, each isflanked by the parental viruses SP-N and LP-B. All of the recombinants shown possess a p30that comigrates with the p30 of SP-N. Figure 5aand b shows gels on which virion proteins wereloaded at a concentration optimal for visualiz-ing p15 bands. Of the recombinants shown, two(Fig. 5a, track 9, and Fig. 5b, tracks 2 and 4)possess a p15 that migrates like the p15 of LP-B.Figure 6 is an autoradiogram showing the

gp7O's of six recombinants. Five of these recom-_ 0 D 70 binants possess a gp7O that migrates like the

gp7O of LP-B; one (track 4) possesses a gp7Owhose electrophoretic mobility is like that ofSP-N gp7O. Some variation in the width of gp7Obands has been noted, and sometimes the resultof this variation is to give the impression thatrecombinant virus gp70's differ slightly fromthose of either SP-N or LP-B; however, we donot know if this difference is significant.Table 1 is a summary ofdata from this type of

analysis of all 21 recombinants. Of the 21 vi-- p30 ruses, 16 possess a gp7O that migrates like the

gp7O of LP-B and 2 out of 21 possess a p15 thatcomigrates with p15 of LP-B, whereas nonepossesses a p30 that migrates with the p30 ofLP-B. All of the remaining proteins (with theexception of the gp7O of recombinant 5-1, whose

40 -0-NW4NO- p15

1 2 3 4FIG. 2. Comparison ofgp7O protein of SP-N and

LP-B viruses. Autoradiogram of '4C-labeled virionproteins; 8 to 20% slab gel. Samples are as follows: 1and 3, LP-B; 2 and 4, SP-N.

predicted, we could now ask whether clonal iso-lates of XLP-N virus possessed p15, p30, andgp7O proteins with the electrophoretic mobilitycharacteristic of the corresponding proteins oftheir N- or B-tropic virus parent. Sc-1 cells wereinfected with each of 21 clonal isolates of XLP-N derived from seven independent crosses ofSP-N and LP-B. Much of the information per-taining to the origin of these viruses has beenreported (10, 12). It is summarized in Fig. 3.Figure 4 is a gel on which virion proteins were

loaded at a concentration optimal for visualiz-

Coinfect Sc-i cells (8) with SP-N and LP-B virusesJ, Use progeny' to infect

NIH/3T3 cellsHarvest progeny2 3days later. Infect

NI /3T3 cellsHarvest progeny3 3days later. Determineend point titer by in-fection of

NIH/3T3 cellsHarvest progeny4 fromend point infection.

Cross (1) Plaque purify XLP-N from progeny4 toobtain 1-Pl.

(2) Microtiter clone XLP-N from progeny3 toobtain 2-1, 2-2.

(3) Microtiter clone XLP-N from progeny3 toobtain 3-1, 3-2.

(4) Microtiter clone XLP-N from progeny3 toobtain 4-1.

(5) Microtiter clone XLP-N from progreny3 toobtain 5-1, 5-2, 5-3, 5-4, 5-5.

(6) Microtiter clone XLP-N from progeny3 toobtain 6-1, 6-2, 6-3.

(7) Plaque purify XLP-N from progeny' toobtain 7-Pl, 7-P2, 7-P3, 7-P4, 7-P5, 7-P6,7-P7.

FIG. 3. Origin ofXLP-N viruses.

J. VIROL.

VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 703

B R N R B R N R B R N R

- - - -_- -. - -p30

1 2 3 4 5 6 7 8 9 10 11 12FIG. 4. Comparison ofp30 protein of SP-N, LP-B, and recombinants. An 8 to 24% slab gel stained with

Coomassie brilliant blue. Samples are as follows: 1, LP-B; 2, 2-1; 3, SP-N; 4, 2-2; 5, LP-B; 6, 1-PJ; 7, SP-N; 8,4-1; 9, LP-B; 10, 7-P2; 11, SP-N; 12, 5-4.

electrophoretic mobility has not been deter-mined) appear to migrate with those of SP-N.The last column in the table shows the re-

sults of analysis of the parental and recombi-nant viruses for the ability to induce the G1xantigen, an antigenic determinant believed toreside on the gp7O of some murine leukemiaviruses and that is present on cells infected bythe N-tropic (G+ ) but not the B-tropic (G- )parent of the recombinants. These data aretaken from Hopkins et al. (10). XLP-N virusesthat are G& possess a gp7O that appears tomigrate with SP-N virus gp7O, whereas thegp7O of G1x XLP-N viruses migrates like thegp7O of LP-B virus. This result is consistentwith evidence suggesting that Gjx resides ongp70 (28).

DISCUSSIONWe have used SDS-polyacrylamide gel elec-

trophoresis to examine three virion proteins ofSP-N, an N-tropic virus that makes small XCplaques, LP-B, a B-tropic virus that makeslarge XC plaques, and 21 large XC plaque-forming N-tropic recombinants, designated

XLP-N viruses, between SP-N and LP-B. ThatXLP-N viruses are recombinants was sug-gested from their biological properties (12) andfrom antigenic analysis (10). Since Famulariand Fleissner (personal communication) hadshown, and we have confirmed here, that thep15, p30, and gp7O proteins of N- and B-tropicviruses of BALB/c each differ in electrophoreticmobility on SDS-polyacrylamide gels, we ex-amined these proteins in XLP-N viruses to de-termine whether the recombinants inheritedboth SP-N- and LP-B-like proteins. We havefound that some XLP-N viruses possess a p15and/or gp70 with the electrophoretic mobilitycharacteristic of the corresponding proteins oftheir B-tropic virus parent.A simple interpretation of these results is

that XLP-N viruses that possess a p15 or gp7Othat comigrates with p15 or gp7O of LP-B inher-ited the corresponding genes from LP-B and,similarly, that migration of an XLP-N virusp15, p30, or gp70 with the corresponding pro-teins of SP-N implies inheritance of the corre-sponding genes from SP-N. However, since p15and p30 are cleaved from a precursor protein

VOL. 23, 1977

C mM B R N R B R N R B

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2 3 4 5 6FIG. 5. Comparison ofp15 protein of SP-N, LP-B, and recombinants. An 8 to 24% slab gel stained with

Coomassie brilliant blue. Samples are as follows: Gel a-1, marker mix as in Fig. 1; 2, LP-B; 3, 5-2; 4, SP-N;5, 5-3; 6, LP-B; 7, 5-4; 8, SP-N; 9, 5-5; 10, LP-B. For gel b: 1, LP-B (the band below p30 in LP-B is acontaminating cell protein present in this particular preparation of virus); 2, 5-5; 3, LP-B; 4, 6-2; 5, LP-B; 6,6-3; 7, LP-B.

704

VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 705

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-p30

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6 7 8 9 10 11 12 13 14FIG. 6. Comparison of the gp7O protein ofSP-N, LP-B, and recombinants. Autoradiogram of '4C-labeled

virion proteins; 8 to 20% slab gel. Samples are as follows: 1, LP-B; 2, 1-PZ; 3, SP-N; 4, 5-4; 5, LP-B; 6, 7-6; 7,SP-N; 8, SP-N; 9, 6-1; 10, LP-B; 11, 3-2; 12, SP-N; 13, 6-3; 14, LP-B.

coded for by the so-called gag gene (23, 29), andsince the "rules" governing this cleavage arenot known, other interpretations can be imag-ined, and studies on the primary structure ofthe proteins involved would be necessary toconfirm this simple interpretation. In any case,that XLP-N viruses inherit proteins with theelectrophoretic mobility characteristic of thecorresponding proteins of LP-B virus wouldseem to provide evidence that these viruseshave inherited portions of the LP-B virus ge-nome that determine the molecular parametersof those proteins. Thus, the results presentedprovide evidence that XLP-N viruses are ge-netic recombinants.Of the 21 recombinants studied, 16 possess a

gp7O that comigrates with the gp70 of LP-B.Only 2 ofthe 21 recombinants possess a p15 that

comigrates with the p15 of LP-B, and nonepossesses a p30 with the electrophoretic mobil-ity of LP-B p30. Recombinants obtained fromany one cross could have arisen from a singlerecombination event, unless they have beenshown to differ in their virion proteins. Thus,the minimum number of independent recombi-nants studied is 11, ofwhich 7 have a gp7O withthe electrophoretic mobility characteristic ofLP-B virus gp7O and 2 have a p15 that comi-grates with p15 of LP-B.

If one makes the assumption that comigra-tion ofXLP-N virus proteins with those ofSP-Nor LP-B implies inheritance of the corre-sponding gene from the N- or B-tropic parent,if one assumes that recombination is equallyprobable along the genome, and if one alsoassumes that the only selection pressures oper-

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Omilp -4

Owe am* one

706 SCHINDLER, HYNES, AND HOPKINS

TABLE 1. Results ofanalysis ofthree virion proteinsofparental and recombinant viruses by SDS-

polyacrylamide gel electrophoresis

Electrophoretic mobility of:

Virus p15 p30 gp7O G,,a

SP-N Slowerb Faster Faster +LP-B Faster Slower Slower

1-P1 Like N Like N Like B2-1 N N N +2-2 N N B -3-1 N N B -3-2 N N B -4-1 N N N +5-1 N N -c +5-2 N N B -5-3 N N B -5-4 N N N +5-5 B N B -6-1 N N B -6-2 B N N +6-3 N N B -7-P1 N N B -7-P2 N N B -7-P3 N N B -7-P4 N N B -7-P5 N N B -7-P6 N N B -7-P7 N N B -

a + or - refers to the ability or inability, respectively, ofSc-1 cells infected by the particular virus to absorb cytotoxicantibody to Gx. These data are taken from Hopkins et al.(10).

b"Slower" or "faster" refers to the relative electropho-retic mobilities of SP-N and LP-B virus p15, p30, and gp7Oproteins.e- Electrophoretic mobility of the gp7O of recombinant

5-1 has not yet been determined.

ating during isolation of the XLP-N viruseswere for N-tropism and large XC plaque mor-phology, then the data presented above haveseveral implications of biological interest. (i)That an N-tropic virus may posses a p15 or gp70protein like that ofa B-tropic virus implies thattropism does not reside in either of these pro-teins (10, 13, 17).

(ii) That some XLP-N viruses, all of whichmake large XC plaques, appear to possess agp7O derived from SP-N, which makes smallXC plaques, suggests that large XC plaquemorphology is not determined solely (if at all)by gp7O (10). However, that so many recombi-nants inherited gp70 from LP-B might suggestthat the determinants(s) of plaque morphologylies near the gene coding for gp7O. (Note thatthese results do not exclude the possibility thatgp70 is involved in the formation of XC cellsyncytia.)

(iii) One interpretation of the observationthat all of the recombinants studied so far in-herited a p30 that comigrates with p30 of SP-N

is that N-tropism might be determined by agene that is closely linked to p30. We haveobtained other evidence consistent with thepossibility that p30 itself, or a gene closelylinked to p30, might be a determinant ofN- andB-tropism: murine leukemia virus variantsthat grow equally well on N- and B-type mousecells can be obtained by forced passage of a B-tropic virus through N-type cells in vitro. Theresulting virus is called NB-tropic (1, 11). EightNB-tropic viruses independently derived fromthe B-tropic virus of BALB/c by serial passageon NIH Swiss mouse cells have been found topossess a p30 with altered electrophoretic mo-bility on SDS gels (11). One of the eight pos-sessed an altered p15 in addition (unpublisheddata). Both the apparent linkage of p30 and N-tropism in the present study and the observa-tion ofan altered- p30 in NB-tropic viruses couldbe explained in a number of ways that areunrelated or only indirectly related to the tro-pism of the viruses (see 21). However, it isinteresting to note that the possibility that aprotein present in virions may be a determi-nant of N- or B-tropism is implied by the stud-ies of Rein et al. (24) and Bassin et al. (4).

Since RNA tumor viruses are polyploid andcan form heterozygotes (30), an alternative ex-planation to recombination for the results wehave presented might be that XLP-N virusesare stable heterozygotes of SP-N and LP-B ge-nomes and selectively express, or package,some proteins coded by one genome and somecoded by the other parental genome. Evidencethat XLP-N viruses are recombinants that in-herited only some of their genes from their Nvirus parent and others from their B virus par-ent comes from analysis of the RNase Ti-resist-ant oligonucleotides derived from the RNA ge-nomes of parental and recombinant viruses(Faller and Hopkins, unpublished data). Thisanalysis reveals great similarity between thegenomes of the N- and B-tropic viruses derivedfrom BALB/c; of approximately 35 unique oligo-nucleotides generated by RNase T1 cleavageand two-dimensional gel electrophoresis, about30 appear to be shared between the two viruses.However, each parental virus possesses six orseven distinctive oligonucleotide "spots," andthe XLP-N viruses that have been analyzed sofar have been shown to possess different combi-nations ofN and B specific spots. The simplestinterpretation of this result is that XLP-N vi-ruses are genetic recombinants as suggested bythe biological evidence (12), antigenic analysis(10), and the study described above.

ACKNOWLEDGMENTSThese studies are based on observations made by Nancy

Famulari and Erwin Fleissner, Sloan Kettering Institute.

J. VIROL.

VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 707

We thank Dr. Famulari for many helpful discussions andfor communicating unpublished data.

This work was supported by Public Health Servicegrants from the National Cancer Institute (CA 19308 toN.H., CA 17007 to R.H., and CA 14051 to the Center forCancer Research, MIT) and a Public Health Service grantfrom the National Institutes of Health, Biomedical SciencesSupport to MIT (RR 07047 to N.H.).

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