Proc. Natl. Acad. Sci. USAVol. 92, pp. 4828-4832, May 1995Immunology

Mouse mammary tumor viruses with functional superantigengenes are selected during in vivo infection

(viral recombination)

T. V. GOLOVKINA*, J. P. DUDLEYt, A. B. JAFFE*, AND S. R. Ross*t*Department of Microbiology/Cancer Center, University of Pennsylvania, Philadelphia, PA 19104-6142; and tDepartment of Microbiology, University of Texas,Austin, TX 78712

Communicated by Philippa Marrack; Howard Hughes Medical Institute, Denver, CO, February 7, 1995

ABSTRACT Mouse mammary tumor virus (MMTV) en-codes a superantigen that is important for viral infectivity invivo. To determine whether superantigen function was re-quired for infection by milk-borne MMTV, we created HYBPRO/Cla transgenic mice. These mice produced a full-length,packaged viral RNA with a frameshift mutation that causedpremature termination of the superantigen protein. YoungHYB PRO/Cla mice showed no deletion of their cognateV,14+ T cells, although they shed virus in their milLk Thenontransgenic offspring of the HYB PRO/Cla mice wereinfected with this virus, since transgene-specific viral tran-scripts were detected in their mammary glands. Surprisingly,these offspring demonstrated the progressive deletion ofV114+ T cells characteristic of exogenous MMTV(C3H) in-fection. Sequence analysis demonstrated that these newlyacquired viruses had reconstituted superantigen open readingframes resulting from recombination between the HYBPRO/Cla and endogenous Mtv-l proviral RNAs. Thus, thereis selection during the infection process for MMTVs withfunctional superantigen genes.

Mouse mammary tumor virus (MMTV) is a retrovirus that isacquired either through the germline as an endogenous virusor by milk-borne infection (1). Although the ultimate target forMMTV is the mammary gland, cells of the immune system playa role in milk-borne virus infection. MMTV encodes a viralprotein [superantigen (Sag)] in its 3' long terminal repeat(LTR) (2, 3) that functions in milk-borne transmission (4, 5).The virus-encoded Sag protein is presented by antigen-presenting cells (APCs), such as B cells, to T cells bearingspecific 3-chain variable (Vp) regions of the T-cell antigenreceptor. This presentation of Sag causes the proliferation ofspecific Vp-bearing T cells when it is recognized as foreign (2)and deletion of such T cells when it is recognized as self (3).Recent data indicate that the role of Sag is to stimulate divisionand cytokine production by cognate T cells, thereby creatinga reservoir of actively dividing T or bystander B cells suscep-tible to MMTV infection (4-6). These infected cells exist athigh enough levels for a long enough period of time to allowthe mammary gland, in which cells begin to divide under thehormonal stimulation of puberty (at 3-4 weeks of age), tobecome infected.Although the importance of the immune system in the

MMTV life cycle is well known, an absolute requirement forthe sag gene in this cycle has not been established. It is possiblethat there are alternative pathways of MMTV infection thatbypass the need for the Sag protein. To test this, we createdHYB PRO/Cla transgenic mice carrying a new MMTV pro-virus with a mutation that caused premature termination of theMMTV(C3H) Sag. The HYB PRO/Cla transgenic femalesproduced milk-borne virus containing the transgene as the

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viral genomic RNA. Surprisingly, offspring that nursed onthese transgenic animals showed deletion of their cognateVp14+ T cells, with similar kinetics to that caused by wild-typeexogenous MMTV(C3H). Sequence analysis of the LTRs ofthe newly acquired viruses showed that recombination be-tween the transgene and endogenous Mtv-1 proviral RNAcaused reconstitution of the sag open reading frame. Theseresults indicate that there is selective pressure for viruses thatencode functional Sag proteins and suggest that Sag activity isrequired for virus transmission.

MATERIALS AND METHODSPlasmids. The plasmid hybrid MMTV (HYB PRO in this

paper) was a gift from G. M. Shackleford, University ofSouthern California (7) (Fig. 1). The HYB PRO/Cla trans-gene was constructed by partial Cla I digestion of HYB PRO,filling in the 5' overhang with Klenow DNA polymerase, andreligation. The sequence of the mutation created in the 3' LTRis shown in Fig. 1. Although the Cla I digest and fill-in shouldhave generated a 2-bp insertion, only a 1-bp insertion wascreated.

Generation of Transgenic Mice. Female and male C3H/HeN MTV- and C3H/HeN MTV' inbred mice and SWoutbred mice were purchased from the Frederick CancerResearch Facility, Frederick, MD. Transgenic mice bearingthe HYB PRO construct have been described (8). The HYBPRO/Cla transgene was introduced into C3H/HeN MTV-mice by our standard procedure. Transgenic animals wereidentified by Southern blot analysis (8).

Cells and Flow Cytofluorometry. Peripheral blood lympho-cytes were stained with titrated amounts of antibodies [ratanti-CD4 or anti-CD8 labeled with phycoerythrin (GIBCO/BRL) and fluoresceinated rat anti-Vp14 (9) or rat anti-Vp6(PharMingen)] and analyzed on an electronically programma-ble individual cell sorter (Coulter) (4).RNase T1 Protection Analysis. Total RNA (40 ,ug) isolated

from virgin and lactating mammary glands and a probecontaining a region of the 3' LTR of exogenous MMTV(C3H)were used for RNase T1 protection analysis (8).cDNA Library Construction and Screening. RNA was iso-

lated from the virus fraction of milk (10) and a cDNA librarywas made with the SuperScript plasmid system for cDNAsynthesis and plasmid cloning (GIBCO/BRL). The plasmidDNAs of clones that hybridized with a MMTVLTR probe (11)were sequenced.

Sequence Analysis. The plasmids were sequenced with aSequenase kit (United States Biochemical). The primers used(arrows in Fig. 1) were no. 1, 5'-AATTCGGAGAACTCGAC-

Abbreviations: LTR, long terminal repeat; MMTV, mouse mammarytumor virus; RT, reverse transcription.tTo whom reprint requests should be addressed at: Department ofMicrobiology/Cancer Center, University of Pennsylvania, 526 CRB,415 Curie Boulevard, Philadelphia, PA 19104-6142.

4828

Proc. Natl. Acad ScL USA 92 (1995) 4829

R C3H exoI L HYB PRO

Pol

-

1 2 3 4"107 cimTCC TCT TAC M CCO CATCMmTTOT CCTTCA GMATAAGA...

S Y K P H R F C P T E I R

CUT WITH CblFILL4NLIGATE

TCC TCT TAC CCO CAT CeC ATT TTO TCC TTC AGAMT AGA MT MT OC TOAS 8 Y K P H R I V F R N R N K N 8 W

FIG. 1. Map of the HYB PRO construct with the three viraltranscripts. The numbered arrows denote the approximate location ofthe primers used for sequencing of the recombinant viruses. Alsoshown is the Cla I frameshift mutation (underlined) introduced intothe 3' LTR of the HYB PRO/Cla transgene. The boxed sequenceTAA is the premature termination codon. R, EcoRI; C, Cla I.

CTTCC-3' [nt 268-289 in the MMTV LTR (12)]; no. 2,5'-TTGTAAGAGGAAGTTGGCTG-3' (nt 326-307); no. 3,5'-CTCCTTGGTATGGAAAATCTTTCC-3' (nt 894-871);and no. 4, 5'-GGACTGTTGCAAGTTTACTC-3' (nt 1203-1184).Reverse Transcription (RT)-PCR Analysis. Total RNA (20

jig) isolated from lactating mammary gland was reverse tran-scribed with SuperScript II reverse transcriptase in the buffersupplied by the manufacture (GIBCO/BRL) and a (dT)15primer (Promega). One-fourth of each cDNA preparation wasused for PCR with primers specific for exogenousMMTV(C3H) [no. 1 and no. 5 (5'-TAATGTTCTATTAGTC-CAGCCACTGT-3; nt 923-898)] or endogenous and exoge-nous MMTVs [no. 6 (5'-GAGACGCTCAACCTCAATTGA-3'; nt 507-527) and no. 4], as described (6). After PCRamplification, 20 ,lI of each reaction mixture was digested withMun I (GIBCO/BRL). The PCR products were analyzed in1.8% agarose gels.

RESULTS

HYB PRO/Cla Transgenic Females Produce Virus. Wepreviously created a strain of mice, HYB PRO, with a genet-ically engineered MMTV provirus as a transgene in theC3H/HeN MMTV- inbred background (8). This transgeneencodes a Sag protein with the same Vp specificity as exoge-nous MMTV(C3H) virus (Vp14). C3H/HeN MMTV- micewere also used to create transgenic mice carrying the HYBPRO construct with a frameshift mutation at aa 113 thatresulted in premature termination of the Sag protein encodedby the 3' LTR (HYB PRO/Cla) (Fig. 1). Transgenic micemade with this mutated sag gene under the transcriptionalcontrol of the MMTV LTR, but lacking the other viral genes,showed no deletion of their Vp14+ T cells (unpublished data).These results indicated that the Cla I frameshift abolished Sagfunction.Two independent strains ofHYB PRO/Cla transgenic mice,

nos. 15 and 28, were made. All of the transgenic mice were bredto nontransgenic C3H/HeN MTV- mice to generate pedi-grees (Fig. 2). RNA expression analysis was carried out on theheterozygous, transgenic offspring of nontransgenic C3H/HeN female mice mated with transgenic males. A probespecific for the hypervariable region of sag of MMTV(C3H)

FIG. 2. Pedigree of the HYB PRO/Cla transgenic mice. 0, Non-transgenic females; o, nontransgenic male; o, transgenic female; o,transgenic male.

was used for RNase T1 protection assays (Fig. 3). Expressionof the transgene was detected in the mammary glands of bothstrains of transgenic HYB PRO/Cla females (shown for strain28; band t of 340 nt in Fig. 3, lane 6). The transgene in both

1 2 3 4 5 6 7 8 9

- 404-327

- 231

- 141

Mtv-1 <

118 -15 107440

340

-> Probe

Full-lengthprotection

FIG. 3. Mice nursed on HYB PRO/Cla transgenic females getinfected with MMTV. RNase protection analysis was performed onRNA isolated from the mammary glands of various lactating andvirgin females (see Fig. 2), using the MMTV(C3H)-specific probe (8,10). Lane 1, tRNA; lanes 2 and 3, lactating mammary glands ofC3H/HeN MMTV+ mice; lane 4, virgin mammary gland from thetransgenic offspring of a HYB PRO/Cla 28 female (mouse B in Fig.2); lane 5, virgin mammary gland from the nontransgenic offspringof a HYB PRO/Cla 28 female (mouse C in Fig. 2); lane 6, lactatingmammary gland from a HYB PRO/Cla 28 female (mouse A in Fig.2); lane 7, lactating mammary gland from the transgenic offspringof a HYB PRO/Cla 28 female (mouse B in Fig. 2); lane 8, lactatingmammary gland from the nontransgenic offspring of a HYB PRO/Cla 28 female (mouse C in Fig. 2); lane 9, lactating mammary glandfrom a C3H/HeN MMTV- female. Diagram shows the probe andthe expected fragments generated in the RNase protection assay. Atleft of the autoradiogram: p, probe (440 nt); t, 340-nt band protectedby the transgene or MMTV(C3H) RNA; Mtv-1, RNA expressedfrom Mtv-1 endogenous locus [results in two protected fragments of118 and 107 nt (black boxes) due to incomplete homology with theMMTV(C3H) probe]. At right: size markers (Taq I fragments ofphage 4x174 DNA).

Mtv-1

gagw~~~~~

Immunology: Golovkina et at

4830 Immunology: Golovkina et al

strains was expressed at about one-tenth the level

endogenous Mtv-1 provirus (bands at 118 and 107 nt).

Histoblot analysis (4) of the newborn offspring

strains of mice was carried out to determine whether

PRO/Cla transgenic mice produced virus. Both HYB PRO/

Cla strains, as well as HYB PRO transgenic females,

into milk, since viral RNA was present in the milk-filled

stomachs of their newborn offspring (unpublished

Therefore, the HYB PRO/Cla transgene was packaged

virions and shed into milk, where it could be acquired

suckling offspring.Virus Shed byHYB PRO/Cla Females Is Infectious.

determine whether the MMTV produced by the HYB PRO/

Cla females was infectious, we examined mammary

from their nontransgenic offspring for expression

integrated exogenous virus. The HYB PRO/Cla-nursed

spring acquired virus, since RNA specific for MMTV(C3H)

was detected in the mammary glands of both their transgenic

and their nontransgenic offspring (Fig. 3, lanes 4, 5,

Thus, the virus produced by the HYB PRO/Cla transgenic

females was infectious.Virus Produced by the EIYB PRO/Cla Transgenic

Regained Vp14-Specific Sag Activity. The results

the preceding section suggested that there was

independent pathway of MMTV infection, that the

retained Sag activity despite the frameshift mutation,

the mutation reverted, resulting in a functional

whether the frameshift mutation inactivated Sag, we

the level of peripheralV.14+ T cells in the HYB PRO/Cla

founder animals or the offspring of transgenic fathers

4A). In contrast to the HYB PRO transgenic mice,

virtually no Vp14+ T cells in their immune repertoire

birth (8), young HYB PRO/Cla 15 and 28 animals (<10

of age) showed no deletion of either CD4+ (shown

15 in Fig. 4A) or CD8+ cognate T cells (unpublished

This was not because of the level of expression

gene; mice with virtually undetectable levels of sag

RNA showed early and complete deletion of

Vp14 T cells (8). A slow progressive loss of Vp144seen as both strains of HYB PRO/Cia mice aged,

that observed in nontransgenic C3H/HeN mice

exogenous MMTV (Fig. 4B). This temporal decline Vpl44T cells suggested that there was production of

wild-type Sag as the HYB PRO/Cia mice aged.

If the Sag activity of MMTV was restored, virus

the V144-specific Sag should be present in

HYB PRO/Cia females. To test this, female PRO/Cia

transgenic mice were mated and the percentage V14+ T

cells was measured in their transgenic and

offspring (Fig. 4B). These offspring showed the

and percent deletion of Vpl44/CD4+ T cells

offspring or mice nursed on C3H/HeN MMTV+

Thus, infectious virus carrying a Sag withVsl44 specificitywas

present in the HYB PRO/Cia offspring.

Sequence Analysis of the Infectious Viruses.

original construct used to make transgenic mice

tion in the sag gene, this mutation was apparently

virus acquired by the HYB PRO/Cia offspring.

mechanism for the regeneration of functional

recombination with an endogenousMtv. Mtv-1

likely partner for recombination with the PRO/Cia

virus, since we previously showed that this provirus

expressed in mammary gland and efficiently

exogenous virus in C3H/HeNM1TV+ mice

the sequences of the MMTV LTRs are

outside the hypervariable region, there are

differences that allow them to be distinguished

(12). For example, nt 378 (C), 394 (G),"a-n-d 4-4-4-(-T-)ar-e s-pe-c-ifilcfor Mtv-1 (Fig. 5). To determine whether functional

created by recombination with Mtv-1, the

12 _

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0

C 5

+

2

OL

5

10

8

'4.

110

6

4

2

10 15 20 25 30 3

5 10 15

15

20

Age, weeks

FIG. 4. (A) Deletion of Vp14-bearing T cells in HYB

PRO/Cia 15, and uninfected C3H/HeN mice. Peripheral

analyzed for the percentages of CD4+Vpl44 and CD4+Vp66Each point represents the average of results for three A,HYB PRO CD4+ Vpl44 ; o, HYB PRO/Cia 15 CD4+ Vpl44 ;;o

PRO/Cia 15 CD4+V,96+; 0, C3H/HeN MTV- CD4+Vpl44.Deletion ofVpl41 T cells in the offspring of the HYB

females. Peripheral T cells were analyzed for the

CD4+V,914+ T cells. Each point represents the average

five to seven mice.o

, C3H/HeN MTV-;°C3H/HeN MTV+; 0,

C3H/HeN nursing on HYB PRO;D, C3H/HeN nursing

PRO/Cia.

LTRs of the viruses acquired by the offspring of

PRO/Cia mice were examined. This analysis also allowed

determine whether recombinant viruses were highly repre-

sented in the milk of HYB PRO/Cia nontransgenic offspring.

A cDNA plasmid library was made from the RNA

pooled virus fraction of milk taken from four nontransgenic

mice that had nursed on HYB PRO/Cia 15 transgenic

(represented by mouse C in Fig. 2). Plasmids from

of colonies that hybridized to a LTR probe were

All of the plasmids had inserts of :1.3 kb, indicating

entire sag coding region was present.

To determine the Vp specificity of the viruses,

plasmids were sequenced with primer 4 (diagrammed

1). Of eight plasmids sequenced, seven had the

variable-region sequence as MMTV(C3H)

data). The remaining cDNA had the same hypervariable

region sequence as Mtv-1. Further sequence

clone showed that it was derived from packaged Mtv-1

Thus, the majority of the viruses transmitted

AT~~~~~~~~~~.1.~~~~~~~~~~I~~~~~~~~~~

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Proc. Natt Acad Sci USA 92 (1995)

AA

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Immunology: Golovkina et aL

C3Iiexo ATCTCTG TGCA.MATTA CMTCTMAC MTCGWA ACTCGACCTT CCTCCTGAGG

C3Hexo ^LACCAC AGCCMC1TC CTCTTACAA CC TTGTCCTrc AGATAGAARld

R'3 ACCR4 .. A. ...C ......... ..........

C3Hexondog.

R2R3R4

C3Hexordog.R2R3R4

C3Hexor7dog.R2R3R4

C3Hexoendog.RlR2R3R4

C3Hexondog.R2R3R4

NIAATGC 1TGICTAAAAA TTATATTMT ACCAATAA CCAATCCMT AGGTAGATrA~ ~~~~~~c......... ...... . . ... . . ... §

~~~~~~~. . . . . . . . . . .. .. ... .. . . .. . . . . . .

~~~~~~~C......... ......G..: . . ...............

flLTrACTA TGTTMGAMAA T6ATCAT1AG TCT1AGTMA CTATTMAC TCAAATTCAGA.. ....... ......A.. ....A..... .........A

....C . ....A..... .........A

AGAAGT...A.C.Tt.... ..... .... .......... .......... Q.p .........

... ....:.... .......... A.^

. ...A.c. . :...... ..T. A: ... ... : ... 4: : ......A1TAGAA T6G6AAMTA MATAGAAAG MAC6CTCM CCT«IAAGUST

.G ......... ..... ..... ..... ..... .... ..... ..... ... ..

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.......... ......... .......... ....................... ...... .................

- - t-- - * ... .............::.:................. . . .... ..........A.... ..... . .. ...C EC. ......

MMAA 6GTGTGCA CCTAGACT TATAMGGGAC CTTACATCTA CAGACCAACA.T.T.. ..C..... G.

. ............ ... ....... ............ ..... ..... ..... ..... ..... .......... .....: ... ....... ......T..CG

FIG. 5. Sequence of the recombinant viruses. Numbering is ac-cording to Brandt-Carlson et aL (12). Brackets denote the recombina-tion junction; single underline, bases unique to Mtv-6; double under-line, bases unique to Mtv-1. exo, Exogenous; endog., endogenous.

PRO/Cla females contained genomic RNAs with Sags en-coded by the transgene.Three additional primers (nos. 1-3 in Fig. 1) were used for

sequencing (Fig. 5). All seven cDNA clones with the trans-gene-specific sag represented recombination events betweenthe endogenous Mtv-1 provirus and the HYB PRO/Cla viralRNA. The viruses could be divided into four different groupsaccording to their recombination breakpoint (R1-R4). Rlrecombinants (two of seven) had a breakpoint between nt 338and 377, since nt 337 was contributed by Mtv-1 sequence andnt 377 by MMTV(C3H). Similarly, R2 recombinants (one ofseven) had a breakpoint between nt 427 and 443, R4 recom-binants (three of seven) between nt 590 and 564, and R3recombinants (one of seven) at nt 580. Because the recombi-nants had Mtv-1 sequence until after the mutated Cla I site inthe transgene, all four types of recombination restored theopen reading frame of the MMTV(C3H) Sag.

Infectious, Recombinant MMTVs Are Selected in the HYBPRO/Cla Offspring. The results described in the precedingsection indicated that recombinant viruses were being selectedin mice that nursed on HYB PRO/Cla females. However, itwas also possible that the recombinant viruses were producedin the HYB PRO/Cla females because they replicated betteror were packaged more efficiently than the virus encoded bythe transgene. We used RT-PCR to examine whether therecombinant viruses were produced in HYB PRO/Cla trans-genic females or their offspring. The primer pairs used did notamplify any endogenous Mtv loci present in the C3H/HeNmouse strain (Fig. 6 Left, lanes 11-13), since the 3' primer (no.5) hybridizes to the hypervariable region of Sag unique toexogenous MMTV(C3H); the 5' primer (no. 1; see Fig. 1)hybridizes to the LTRs of all MMTVs. After RT-PCR, theamplified products were digested with Mun I; this enzyme cutsat position 521 only in the LTR of MMTV(C3H) (Fig. 5),generating fragments of 249 and 407 bp. The recombinationbreakpoints in the viruses isolated from the HYB PRO/Cla-infected nontransgenic mice were located either 5' or 3' of this

Proc. NatL Acad ScL USA 92 (1995) 4831

1 2345678 9

1 2 3 4 5 6 7 8 9 10111213

FIG. 6. Recombinant virus is selected in the HYB PRO/Cia-infected offspring. (Left) RT-PCR assays using primers specific forexogenous MMTV(C3H) and the HYB PRO/Cia transgene followedby Mun I digestion were performed with attn amr lnRNA as described (6). Lanes 2, 5, 8, and 11 are with reversetranscriptase and Mun I; lanes 3. 6. 9. 'and 12 are with reversetranscriptase but without Mun I; lanes 4, 7, 10, and 13 are withoutreverse transcriptase. Lane 1, 1-kb- ladder (GIBCO/BRL); lanes 2-4,RNA from a nontransgenic offspring of a HYB PRO/Cla 15 female(mouse Gin Fig. 2); lanes 5-7, RNA from a HYB PRO/Cla 15 female(mouse A in Fig. 2); lanes 8-10, RNA fromia. transgenic offspring ofHYB PRO/Cla 15 (mouse B in Fig. 2) (mice B and Care the offspringof mouse A); lanes 11-13, RNA from a nontransgenic C3H/HeNmouse. (Right) RT-PCR was performed on the same RNAs as in Left,but with primers 6 and 4, which amplify endogenous and exogenousMMTVs (see Materials and Methods). Lanes 2, 4, 6, and 8, no reversetranscriptase; lanes 3, 5, 7 and 9, with reverse transcriptase. Lane 1,1-kb ladder; lanes 2 and 3, RNA from mouse C; lanes 4 and 5, RNAfrom mouse A; lanes 6 and 7, RNA from mouse B; lanes 8 and 9, RNAfrom an uninfected C3H/HeN mouse.

site (Fig. 5). Since the primers used in' this study could amplifyDNA as well as RNA, the RNAs were also subjected to RT inthe absence of reverse transcriptase, as a negative control. Inaddition, a primer pair that amplified all'MMTVs was used toverify the integrity of the RNA (Fig. 6 Right).When this analysis was carried out with RNA isolated from

the lactating mammary gland of a HYB PRO/Cia 15 trans-genic female (Fig. 6 Left, lanes 5-7; mouse A in Fig. 2) or hertransgenic offspring (lanes 8-10; m'ouse B in Fig. 2), all of theamplified product was digested with Mun I (compare lane 6with lane S and lane 9 with lane 8). Thus, all of the virusproduced by the transgenic HYB PRO/Cia 15 female wasderived from the transgene and there was no evidence ofrecombinant virus production in the absen'ce of selection fora functional Sag. In contrast, when this experiment wasperformed on RNA isolated from' the n'ontransgenic offspringof the HYB PRO/Cia 15 mouse, -5O% of the amplificationproduct was digested with Mun I (lane 2; mouse C in Fig. 2).This is similar to what was seen in the sequencing analysis,where the recombination breakpoints occurred upstream anddownstream of the Mun I site in an approximately 1:1 ratio.That recombinant virus was only found in the nontransgenicoffspring of the HYB PRO/Cia mice indicates that onlyMMTVs with functional sag genes can be propagated.

DISCUSSIONRetroviruses are subject to recombination with other retrovi-ral genomes packaged i-n the same viral particle because thereverse transcriptase can switch templates during replication(13, 14). Copackaging of different genomes occurs when morethan one exogenous virus infects the same cell or when anendogenous virus is expressed in the presence of an infectiousvirus (10, 15). The newly generated recombinants will bepreferentially replicated if they confer a selective advantageover the parental viruses. For example, in the case of poly-tropic murine leukemia viruses, recombination is required for

4832 Immunology: Golovkina et at

the generation of a transforming virus (16). Moreover, recom-bination probably plays an important role in the generation ofpathogenic variants of human immunodeficiency virus (17).MMTV is found as an endogenous virus in the genomes of

all commonly used inbred mice (18) and many feral mice(19-21). Many, but not all, of the endogenous Mtv provirusesare expressed in the mammary gland (10, 22) and thereforecould be copackaged with exogenous virus, where they thenwould participate in the generation of recombinant virus. If theproduct of the recombination had an advantage for viralreplication or transmission, such viruses would be amplifiedselectively and passed on to subsequent generations.

In the work presented here, we determined whether afunctional product of the MMTV sag gene would be selectedduring viral infection, by constructing a viral genome that hada Sag frameshift mutation. We found that infectious virus wastransmitted by transgenic females containing this constructand that Sag function was restored by recombination of theHYB PRO/Cla with the Mtv-1 endogenous virus. We recentlyshowed that there is packaging of the endogenous Mtv-l viralRNA, which is abundantly expressed in the lactating mammarygland of C3H/HeN mice, in the presence of MMTV(C3H)infection (10). Thus, it is not surprising that all of the recom-binant viruses identified here had Mtv-1 as one of theirparental viruses.

It is clear that there was strong selection for infectiousviruses that regenerated a functional sag gene. Since we couldnot detect recombinant viral RNA in the transgenic HYBPRO/Cla females, the predominant form of the virus theyshed into milk was derived from the transgene. In contrast,seven of the eight milk-borne viruses isolated from theirnontransgenic offspring were derived from recombinationbetween the HYB PRO/Cla and Mtv-1 genomes and had areconstituted Sag open reading frame. Either the recombinantvirus was generated in the HYB PRO/Cla transgenic femalesat levels below our detection and then selected in the non-transgenic offspring or the recombination occurred in theoffspring because they were infected with copackaged trans-gene and Mtv-1 RNA. That we did see slow deletion of Vp14+T cells in the HYB PRO/Cla mice indicates that somerecombinatiori took place in these animals. In any case, if therewere an alternative pathway of MMTV infection that did notrequire Sag activity, the parental HYB PRO/Cla virus retain-ing the frameshift mutation also should have been transmittedto offspring, since it was the predominant species of virus shedinto milk. Since only the repaired virus was found, it appearsthat Sag activity is required for viral transmission.

Several groups have identified novel exogenous MMTVsthat encode Sag proteins of different V, specificities (23-25),and it was hypothesized that at least one of these may havearisen by recombination between an exogenous virus (SW21)and an endogenous virus (Mtv-6) (24). Moreover, sequenceanalysis of the various endogenous and exogenous MMTVsindicates that some of these viruses were created by recom-bination (12). We also proposed that copackaging of heterol-ogous RNAs and recombination might broaden the host rangeof MMTV (10). The V3 repertoire of outbred mice is deter-mined in part by the endogenous Mtv proviruses present intheir genomes (26), and exogenous virus can infect only thoseanimals which do not have endogenous counterparts with thesame Sag specificity (4, 5). The endogenousMtv proviruses will

segregate in outbred mice, leaving offspring with susceptibilityto different exogenous MMTVs than their parents. Copack-aging of exogenous and endogenous MMTVs must lead torecombinant viruses that are selected and amplified in micethat retain T cells with ,B chains that interact with the Sagsencoded in such viruses.

We thank A. V. Chervonsky and members of our laboratory forhelpful discussions. We gratefully acknowledge the expertise andequipment provided by the Flow Cytometry Laboratory of the Re-search Resources Center of the University of Illinois of Chicago.T.V.G. is a Cancer Research Institute Fellow. This work was supportedby Public Health Service Grants CA45954 and CA52646 from theNational Cancer Institute.

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