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

Mouse mammary tumor viruses with functional superantigen genes 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- viral genomic RNA. Surprisingly, offspring that nursed on codes a superantigen that is important for viral infectivity in these transgenic animals showed deletion of their cognate vivo. To determine whether superantigen function was re- Vp14+ T cells, with similar kinetics to that caused by wild-type quired for infection by milk-borne MMTV, we created HYB exogenous MMTV(C3H). Sequence analysis of the LTRs of PRO/Cla transgenic mice. These mice produced a full-length, the newly acquired viruses showed that recombination be- packaged viral RNA with a frameshift mutation that caused tween the transgene and endogenous Mtv-1 proviral RNA premature termination of the superantigen protein. Young caused reconstitution of the sag open reading frame. These HYB PRO/Cla mice showed no deletion of their cognate results indicate that there is selective pressure for viruses that V,14+ T cells, although they shed virus in their milLk The encode functional Sag proteins and suggest that Sag activity is nontransgenic offspring of the HYB PRO/Cla mice were required for virus transmission. infected with this virus, since transgene-specific viral tran- scripts were detected in their mammary glands. Surprisingly, these offspring demonstrated the progressive deletion of MATERIALS AND METHODS V114+ T cells characteristic of exogenous MMTV(C3H) in- Plasmids. The plasmid hybrid MMTV (HYB PRO in this fection. Sequence analysis demonstrated that these newly paper) was a gift from G. M. Shackleford, University of acquired viruses had reconstituted superantigen open reading Southern California (7) (Fig. 1). The HYB PRO/Cla trans- frames resulting from recombination between the HYB gene was constructed by partial Cla I digestion of HYB PRO, PRO/Cla and endogenous Mtv-l proviral RNAs. Thus, there filling in the 5' overhang with Klenow DNA polymerase, and is selection during the infection process for MMTVs with religation. The sequence of the mutation created in the 3' LTR functional superantigen genes. is shown in Fig. 1. Although the Cla I digest and fill-in should have generated a 2-bp insertion, only a 1-bp insertion was Mouse mammary tumor virus (MMTV) is a retrovirus that is created. acquired either through the germline as an endogenous virus Generation of Transgenic Mice. Female and male C3H/ or by milk-borne infection (1). Although the ultimate target for HeN MTV- and C3H/HeN MTV' inbred mice and SW MMTV is the mammary gland, cells of the immune system play outbred mice were purchased from the Frederick Cancer a role in milk-borne virus infection. MMTV encodes a viral Research Facility, Frederick, MD. Transgenic mice bearing protein [superantigen (Sag)] in its 3' long terminal repeat the HYB PRO construct have been described (8). The HYB (LTR) (2, 3) that functions in milk-borne transmission (4, 5). PRO/Cla transgene was introduced into C3H/HeN MTV- The virus-encoded Sag protein is presented by antigen- mice by our standard procedure. Transgenic animals were presenting cells (APCs), such as B cells, to T cells bearing identified by Southern blot analysis (8). specific 3-chain variable (Vp) regions of the T-cell antigen Cells and Flow Cytofluorometry. Peripheral blood lympho- receptor. This presentation of Sag causes the proliferation of cytes were stained with titrated amounts of antibodies [rat specific Vp-bearing T cells when it is recognized as foreign (2) anti-CD4 or anti-CD8 labeled with phycoerythrin (GIBCO/ and deletion of such T cells when it is recognized as self (3). BRL) and fluoresceinated rat anti-Vp14 (9) or rat anti-Vp6 Recent data indicate that the role of Sag is to stimulate division (PharMingen)] and analyzed on an electronically programma- and cytokine production by cognate T cells, thereby creating ble individual cell sorter (Coulter) (4). a reservoir of actively dividing T or bystander B cells suscep- RNase T1 Protection Analysis. Total RNA (40 ,ug) isolated tible to MMTV infection (4-6). These infected cells exist at from virgin and lactating mammary glands and a probe high enough levels for a long enough period of time to allow containing a region of the 3' LTR of exogenous MMTV(C3H) the mammary gland, in which cells begin to divide under the were used for RNase T1 protection analysis (8). hormonal stimulation of puberty (at 3-4 weeks of age), to cDNA Library Construction and Screening. RNA was iso- become infected. lated from the virus fraction of milk (10) and a cDNA library Although the importance of the immune system in the was made with the SuperScript plasmid system for cDNA MMTV life cycle is well known, an absolute requirement for and the sag gene in this cycle has not been established. It is possible synthesis plasmid cloning (GIBCO/BRL). The plasmid that there are alternative pathways of MMTV infection that DNAs ofclones that hybridized with a MMTV LTR probe (11) bypass the need for the Sag protein. To test this, we created were sequenced. HYB PRO/Cla transgenic mice carrying a new MMTV pro- Sequence Analysis. The plasmids were sequenced with a virus with a mutation that caused premature termination ofthe Sequenase kit (United States Biochemical). The primers used MMTV(C3H) Sag. The HYB PRO/Cla transgenic females (arrows in Fig. 1) were no. 1, 5'-AATTCGGAGAACTCGAC- produced milk-borne virus containing the transgene as the Abbreviations: LTR, long terminal repeat; MMTV, mouse mammary tumor virus; RT, reverse transcription. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Microbiology/Cancer Center, University of Pennsylvania, 526 CRB, accordance with 18 U.S.C. §1734 solely to indicate this fact. 415 Curie Boulevard, Philadelphia, PA 19104-6142. 4828 Downloaded by guest on September 26, 2021 Immunology: Golovkina et at Proc. Natl. Acad ScL USA 92 (1995) 4829

Mtv-1 R C3H exo I L HYB PRO gag Pol w~~~~~ -

1 2 3 4 "107 cim TCC TCT TAC M CCO CAT CMmTTOT CCT TCA GMATA AGA...

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

CUT WITH Cbl FILL4N FIG. 2. Pedigree of the HYB PRO/Cla transgenic mice. 0, Non- LIGATE transgenic females; o, nontransgenic male; o, transgenic female; o, transgenic male. TCC TCT TAC CCO CAT CeC ATT TTO TCC TTC AGAM T AGA MT MT OC TOA S 8 Y K P H R I V F R N R NKN 8 W was used for RNase T1 protection assays (Fig. 3). Expression FIG. 1. Map of the HYB PRO construct with the three viral of the transgene was detected in the mammary glands of both transcripts. The numbered arrows denote the approximate location of strains of transgenic HYB PRO/Cla females (shown for strain the primers used for sequencing of the recombinant viruses. Also t of 340 nt shown is the Cla I frameshift mutation (underlined) introduced into 28; band in Fig. 3, lane 6). The transgene in both the 3' LTR of the HYB PRO/Cla transgene. The boxed sequence 1 2 3 4 5 6 7 8 9 TAA is the premature termination codon. R, EcoRI; C, Cla I. - 404 CTTCC-3' [nt 268-289 in the MMTV LTR (12)]; no. 2, -327 5'-TTGTAAGAGGAAGTTGGCTG-3' (nt 326-307); no. 3, 5'-CTCCTTGGTATGGAAAATCTTTCC-3' (nt 894-871); - 231 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- - 141 scribed with SuperScript II reverse transcriptase in the buffer supplied by the manufacture (GIBCO/BRL) and a (dT)15 Mtv-1 < primer (Promega). One-fourth of each cDNA preparation was used for PCR with primers specific for exogenous MMTV(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 PCR amplification, 20 ,lI of each reaction mixture was digested with Mun I (GIBCO/BRL). The PCR products were analyzed in 1.8% agarose gels. 118 -15 107 440 -> Probe RESULTS HYB PRO/Cla Transgenic Females Produce Virus. We 340 Full-length protection previously created a strain of mice, HYB PRO, with a genet- ically engineered MMTV provirus as a transgene in the FIG. 3. Mice nursed on HYB PRO/Cla transgenic females get C3H/HeN MMTV- inbred background (8). This transgene infected with MMTV. RNase protection analysis was performed on encodes a Sag protein with the same Vp specificity as exoge- RNA isolated from the mammary glands of various lactating and nous MMTV(C3H) virus (Vp14). C3H/HeN MMTV- mice virgin females (see Fig. 2), using the MMTV(C3H)-specific probe (8, were also used to create transgenic mice carrying the HYB 10). Lane 1, tRNA; lanes 2 and 3, lactating mammary glands of PRO construct with a frameshift mutation at aa 113 that C3H/HeN MMTV+ mice; lane 4, virgin mammary gland from the resulted in premature termination of the Sag protein encoded transgenic offspring of a HYB PRO/Cla 28 female (mouse B in Fig. by the 3' LTR (HYB PRO/Cla) (Fig. 2); lane 5, virgin mammary gland from the nontransgenic offspring 1). Transgenic mice of a HYB PRO/Cla 28 female (mouse C in Fig. 2); lane 6, lactating made with this mutated sag gene under the transcriptional mammary gland from a HYB PRO/Cla 28 female (mouse A in Fig. control of the MMTV LTR, but lacking the other viral genes, 2); lane 7, lactating mammary gland from the transgenic offspring showed no deletion of their Vp14+ T cells (unpublished data). of a HYB PRO/Cla 28 female (mouse B in Fig. 2); lane 8, lactating These results indicated that the Cla I frameshift abolished Sag mammary gland from the nontransgenic offspring of a HYB PRO/ function. Cla 28 female (mouse C in Fig. 2); lane 9, lactating mammary gland Two independent strains of HYB PRO/Cla transgenic mice, from a C3H/HeN MMTV- female. Diagram shows the probe and nos. 15 and 28, were made. All ofthe transgenic mice were bred the expected fragments generated in the RNase protection assay. At to nontransgenic C3H/HeN MTV- mice to generate pedi- left of the autoradiogram: p, probe (440 nt); t, 340-nt band protected by the transgene or MMTV(C3H) RNA; Mtv-1, RNA expressed grees (Fig. 2). RNA expression analysis was carried out on the from Mtv-1 endogenous locus [results in two protected fragments of heterozygous, transgenic offspring of nontransgenic C3H/ 118 and 107 nt (black boxes) due to incomplete homology with the HeN female mice mated with transgenic males. A probe MMTV(C3H) probe]. At right: size markers (Taq I fragments of specific for the hypervariable region of sag of MMTV(C3H) phage 4x174 DNA). Downloaded by guest on September 26, 2021 4830 Immunology: Golovkina et al Proc. Natt Acad Sci USA 92 (1995)

strains was expressed at about one-tenth the level 12 _

at 118 and 107 endogenous Mtv-1 provirus (bands nt). A Histoblot analysis (4) of the newborn offspring T~~~~~~~~~~ strains of mice was carried out to determine whether "O 10 .1.~~~~~~~~~~I~~~~~~~~~~ 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 O 8 0 stomachs of their newborn offspring (unpublished L.. Therefore, the HYB PRO/Cla transgene was packaged

C virions and shed into milk, where it could be acquired 5 suckling offspring. 0

Is Infectious. Virus Shed byHYB PRO/Cla Females +

determine whether the MMTV produced by the HYB PRO/ 2 Cla females was infectious, we examined mammary

from their nontransgenic offspring for expression A -r O a A_A A e . HYB PRO/Cla-nursed integrated exogenous virus. The L

spring acquired virus, since RNA specific for MMTV(C3H) 5 10 15 20 25 30 3 15 was detected in the mammary glands of both their transgenic

and their nontransgenic offspring (Fig. 3, lanes 4, 5, 10 r- Thus, the virus produced by the HYB PRO/Cla transgenic B females was infectious. Virus Produced by the EIYB PRO/Cla Transgenic

Regained Vp14-Specific Sag Activity. The results 8 the preceding section suggested that there was

independent pathway of MMTV infection, that the '4.

retained Sag activity despite the frameshift mutation, the mutation reverted, resulting in a functional 6 whether the frameshift mutation inactivated Sag, we

the level of peripheralV.14+ T cells in the HYB PRO/Cla 110 founder animals or the offspring of transgenic fathers

4A). In contrast to the HYB PRO transgenic mice, T1 4 virtually no Vp14+ T cells in their immune repertoire T birth (8), young HYB PRO/Cla 15 and 28 animals (<10 of age) showed no deletion of either CD4+ (shown I~~~~~~~~I 15 in Fig. 4A) or CD8+ cognate T cells (unpublished 2

This was not because of the level of expression 5 10 1 5 20

gene; mice with virtually undetectable levels of sag Age, weeks RNA showed early and complete deletion of Vp14 T cells (8). A slow progressive loss of Vp144 FIG. 4. (A) Deletion of Vp14-bearing T cells in HYB seen as both strains of HYB PRO/Cia mice aged, PRO/Cia 15, and uninfected C3H/HeN mice. Peripheral that observed in nontransgenic C3H/HeN mice analyzed for the percentages of CD4+Vpl44 and CD4+Vp66

exogenous 4B). This temporal decline three MMTV (Fig. Vpl44 Each point represents the average of results for A, T cells suggested that there was production of HYB PRO CD4+ Vpl44 ; o, HYB PRO/Cia 15 CD4+ Vpl44;o ; PRO/Cia 15 0, C3H/HeN MTV- wild-type Sag as the HYB PRO/Cia mice aged. CD4+V,96+; CD4+Vpl44. Deletion of T cells in the offspring of the HYB If the Sag activity of MMTV was restored, virus Vpl41

females. Peripheral T cells were analyzed for the the V144-specific Sag should be present in CD4+V,914+ T cells. Each point represents the average To test this, female o HYB PRO/Cia females. PRO/Cia ° five to seven mice. , C3H/HeN MTV-; C3H/HeN MTV+; 0, transgenic mice were mated and the percentage V14+ T C3H/HeN nursing on HYB PRO;D, C3H/HeN nursing cells was measured in their transgenic and PRO/Cia.

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

and percent deletion of T cells Vpl44/CD4+ LTRs of the viruses acquired by the offspring of offspring or mice nursed on C3H/HeN MMTV+ PRO/Cia mice were examined. This analysis also allowed

Thus, infectious virus carrying a Sag with specificitywas Vsl44 determine whether recombinant viruses were highly repre- present in the HYB PRO/Cia offspring. sented in the milk of HYB PRO/Cia nontransgenic offspring. Sequence Analysis of the Infectious Viruses. A cDNA plasmid library was made from the RNA original construct used to make transgenic mice pooled virus fraction of milk taken from four nontransgenic tion in the sag gene, this mutation was apparently mice that had nursed on HYB PRO/Cia 15 transgenic virus acquired by the HYB PRO/Cia offspring.

(represented by mouse C in Fig. 2). Plasmids from mechanism for the regeneration of functional of colonies that hybridized to a LTR probe were recombination with an endogenousMtv. Mtv-1 All of the plasmids had inserts of :1.3 kb, indicating likely partner for recombination with the PRO/Cia entire sag coding region was present. virus, since we previously showed that this provirus

specificity of the viruses, expressed in mammary gland and efficiently To determine the Vp

primer 4 (diagrammed exogenous virus in C3H/HeNM1TV+ mice plasmids were sequenced with

the sequences of the MMTV LTRs are 1). Of eight plasmids sequenced, seven had the

outside the hypervariable region, there are variable-region sequence as MMTV(C3H) that allow them to be distinguished differences data). The remaining cDNA had the same hypervariable (12). For example, nt 378 (C), 394 (G),"a-n-d 4-4-4-(-T-)ar-e s-pe-c-ifilc region sequence as Mtv-1. Further sequence

whether functional for Mtv-1 (Fig. 5). To determine clone showed that it was derived from packaged Mtv-1

created by recombination with the Mtv-1, Thus, the majority of the viruses transmitted Downloaded by guest on September 26, 2021 Immunology: Golovkina et aL Proc. NatL Acad ScL USA 92 (1995) 4831

1 2345678 9 C3Iiexo ATCTCTG TGCA.MATTA CMTCTMAC MTCGWA ACTCGACCTT CCTCCTGAGG

C3Hexo ^LACCAC AGCCMC1TC CTCTTACAA CC TTGTCCTrc AGATAGAA Rld 1 2 3 4 5 6 7 8 9 10111213 R'3 ACC R4 .. A. ...C ...... C3Hexo NIAATGC 1TGICTAAAAA TTATATTMT ACCAATAA CCAATCCMT AGGTAGATrA ndog. ~ ~~~~~~c...... § ~~~~~~~...... R2 R3 R4 ~~~~~~~C...... G..: ...... C3Hexo flLTrACTA TGTTMGAMAA T6ATCAT1AG TCT1AGTMA CTATTMAC TCAAATTCAG rdog. A...... A...... A...... A

R2 ....C . .... A ...... A R3 ... R4 ...:...... A.^ FIG. 6. Recombinant virus is selected in the HYB PRO/Cia- ...A.C.Tt...... Q.p ...... AGAAGT infected offspring. (Left) RT-PCR assays using primers specific for C3Hexo A1TAGAA T6G6AAMTA MATAGAAAG MAC6CTCM CCT«IAAGUST and the HYB . ...A.c. . :...... T. A: ...... : ... 4: : ...... exogenous MMTV(C3H) PRO/Cia transgene followed r7dog. by Mun I digestion were performed with attn amr ln R2 .G ...... R3 RNA as described (6). Lanes 2, 5, 8, and 11 are with reverse R4 and Mun lanes 'and 12 are with reverse ...... 3. 6. 9...... transcriptase I; C3Hexo transcriptase but without Mun I; lanes 4, 7, 10, and 13 are without endog...... A.G..T..G. 6.6." C GC.. Rl reverse transcriptase. Lane 1, 1-kb- ladder (GIBCO/BRL); lanes 2-4, R2 RNA from a nontransgenic offspring of a HYB PRO/Cla 15 female R3 - - t-- - * ...... ::.:...... R4 ...... A...... C EC...... (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 of C3Hexo MMAA 6GTGTGCA CCTAGACT TATAMGGGAC CTTACATCTA CAGACCAACA HYB PRO/Cla 15 (mouse B in Fig. 2) (mice B and Care the offspring ndog. .T.T.. ..C..... G. of mouse A); lanes 11-13, RNA from a nontransgenic C3H/HeN R2 ...... : ...... T..CG R3 ...... mouse. (Right) RT-PCR was performed on the same RNAs as in Left, . R4 . but with primers 6 and 4, which amplify endogenous and exogenous MMTVs Materials and Lanes and no reverse FIG. 5. Sequence of the recombinant viruses. is ac- (see Methods). 2, 4, 6, 8, Numbering lanes 5, 7 and with reverse Lane to Brandt-Carlson et aL Brackets denote the transcriptase; 3, 9, transcriptase. 1, cording (12). recombina- 1-kb lanes 2 and RNA from mouse lanes 4 and tion junction; single underline, bases unique to Mtv-6; double under- ladder; 3, C; 5, RNA from mouse A; lanes 6 and RNA from mouse lanes 8 and RNA line, bases unique to Mtv-1. exo, Exogenous; endog., endogenous. 7, B; 9, from an uninfected C3H/HeN mouse. PRO/Cla females contained genomic RNAs with Sags en- site Since the used in' this could coded by the transgene. (Fig. 5). primers study amplify DNA as well as RNA, the RNAs were also subjected to RT in Three additional primers (nos. 1-3 in Fig. 1) were used for the absence of reverse transcriptase, as negative control. In sequencing (Fig. 5). All seven cDNA clones with the trans- a addition, a primer pair that amplified all'MMTVs was used to gene-specific sag represented recombination events between verify the integrity of the RNA (Fig. 6 Right). the endogenous Mtv-1 provirus and the HYB PRO/Cla viral When this was carried out with RNA. The analysis RNA isolated from viruses could be divided into four different groups the mammary of a HYB 15 trans- according to their recombination lactating gland PRO/Cia breakpoint (R1-R4). Rl genic female (Fig. 6 Left, lanes 5-7; mouse A in Fig. 2) or her recombinants (two of seven) had a breakpoint between nt 338 transgenic offspring (lanes 8-10; m'ouse B in Fig. 2), all of the and 377, since nt was sequence 337 contributed by Mtv-1 and amplified product was digested with Mun I (compare lane 6 nt 377 by MMTV(C3H). Similarly, R2 recombinants (one of with lane S and lane 9 with lane 8). Thus, all of the virus seven) had a nt breakpoint between 427 and 443, R4 recom- produced by the transgenic HYB PRO/Cia 15 female was binants (three of seven) between nt 590 and 564, and R3 derived from the transgene and there was no evidence of recombinants (one of seven) at nt 580. Because the recombi- recombinant virus production in the absen'ce of selection for nants had Mtv-1 sequence until after the mutated Cla I site in a functional Sag. In contrast, when this experiment was the transgene, all four types of recombination restored the performed on RNA isolated from' the n'ontransgenic offspring open reading frame of the MMTV(C3H) Sag. of the HYB PRO/Cia 15 mouse, -5O% of the amplification Infectious, Recombinant MMTVs Are Selected in the HYB product was digested with Mun I (lane 2; mouse C in Fig. 2). PRO/Cla Offspring. The results described in the preceding This is similar to what was seen in the sequencing analysis, section indicated that recombinant viruses were being selected where the recombination breakpoints occurred upstream and in mice that nursed on HYB PRO/Cla females. However, it downstream of the Mun I site in an approximately 1:1 ratio. was also possible that the recombinant viruses were produced That recombinant virus was only found in the nontransgenic in the HYB PRO/Cla females because they replicated better offspring of the HYB PRO/Cia mice indicates that only or were packaged more efficiently than the virus encoded by MMTVs with functional sag genes can be propagated. the transgene. We used RT-PCR to examine whether the recombinant viruses were produced in HYB PRO/Cla trans- genic females or their offspring. The primer pairs used did not DISCUSSION amplify any endogenous Mtv loci present in the C3H/HeN Retroviruses are subject to recombination with other retrovi- mouse strain (Fig. 6 Left, lanes 11-13), since the 3' primer (no. ral genomes packaged i-n the same viral particle because the 5) hybridizes to the hypervariable region of Sag unique to reverse transcriptase can switch templates during replication exogenous MMTV(C3H); the 5' primer (no. 1; see Fig. 1) (13, 14). Copackaging of different genomes occurs when more hybridizes to the LTRs of all MMTVs. After RT-PCR, the than one exogenous virus infects the same cell or when an amplified products were digested with Mun I; this enzyme cuts endogenous virus is expressed in the presence of an infectious at position 521 only in the LTR of MMTV(C3H) (Fig. 5), virus (10, 15). The newly generated recombinants will be generating fragments of 249 and 407 bp. The recombination preferentially replicated if they confer a selective advantage breakpoints in the viruses isolated from the HYB PRO/Cla- over the parental viruses. For example, in the case of poly- infected nontransgenic mice were located either 5' or 3' of this tropic murine leukemia viruses, recombination is required for Downloaded by guest on September 26, 2021 4832 Immunology: Golovkina et at Proc. Natl. Acad Sci. USA 92 (1995) the generation of a transforming virus (16). Moreover, recom- segregate in outbred mice, leaving offspring with susceptibility bination probably plays an important role in the generation of to different exogenous MMTVs than their parents. Copack- pathogenic variants of human immunodeficiency virus (17). aging of exogenous and endogenous MMTVs must lead to MMTV is found as an endogenous virus in the genomes of recombinant viruses that are selected and amplified in mice all commonly used inbred mice (18) and many feral mice that retain T cells with ,B chains that interact with the Sags (19-21). Many, but not all, of the endogenous Mtv proviruses encoded in such viruses. are expressed in the mammary gland (10, 22) and therefore could be copackaged with exogenous virus, where they then We thank A. V. Chervonsky and members of our laboratory for would participate in the generation of recombinant virus. If the helpful discussions. We gratefully acknowledge the expertise and product of the recombination had an advantage for viral equipment provided by the Flow Cytometry Laboratory of the Re- be search Resources Center of the University of Illinois of Chicago. replication or transmission, such viruses would amplified T.V.G. is a Cancer Research Institute Fellow. This work was supported selectively and passed on to subsequent generations. by Public Health Service Grants CA45954 and CA52646 from the In the work presented here, we determined whether a National Cancer Institute. functional product of the MMTV sag gene would be selected during viral infection, by constructing a viral genome that had 1. Moore, D. H., Long, C. A., Vaidya, A. B., Sheffield, J. B., Dion, a Sag frameshift mutation. We found that infectious virus was A. S. & Lasfargues, E. Y. (1979) Adv. Cancer Res. 29, 347-418. transmitted by transgenic females containing this construct 2. Choi, Y., Kappler, J. W. & Marrack, P. (1991) Nature (London) and that Sag function was restored by recombination of the 350, 203-207. HYB PRO/Cla with the Mtv-1 endogenous virus. We recently 3. Acha-Orbea, H., Shakhov, A. N., Scarpellino, L., Kolb, E., Mul- showed that there is packaging of the endogenous Mtv-l viral ier, V., Vessaz-Shaw, A., Fuchs, R., Blochlinger, K., Rollini, P., Billotte, J., Sarafidou, M., MacDonald, H. R. & Diggelman, H. RNA, which is abundantly expressed in the lactating mammary (1991) Nature (London) 350, 207-210. gland of C3H/HeN mice, in the presence of MMTV(C3H) 4. Golovkina, T. V., Chervonsky, A., Dudley, J. P. & Ross, S. R. infection (10). Thus, it is not surprising that all of the recom- (1992) Cell 69, 637-645. binant viruses identified here had Mtv-1 as one of their 5. Held, W., Waanders, G., Shakhov, A. N., Scarpellino, L., Acha- parental viruses. Orbea, H. & MacDonald, H. R. (1993) Cell 74, 529-540. It is clear that there was strong selection for infectious 6. Beutner, U., Draus, E., Kitamura, D., Rajewsky, K. & Huber, viruses that regenerated a functional sag gene. Since we could B. T. (1994) J. Exp. Med. 179, 1457-1466. not detect recombinant viral RNA in the transgenic HYB 7. Shackleford, G. M. & Varmus, H. E. (1988) Proc. Natl. Acad. Sci. females, the predominant form of the virus they USA 85, 9655-9659. PRO/Cla 8. Golovkina, T. V., Chervonsky, A., Prescott, J. A., Janeway, C. A. shed into milk was derived from the transgene. In contrast, & Ross, S. R. (1994) J. Exp. Med. 179, 439-446. seven of the eight milk-borne viruses isolated from their 9. Liao, N. S., Maltzman, J. & Raulet, D. H. (1989)1. Exp. Med. 170, nontransgenic offspring were derived from recombination 135-143. between the HYB PRO/Cla and Mtv-1 genomes and had a 10. Golovkina, T. V., Jaffe, A. & Ross, S. R. (1994) J. Virol. 68, reconstituted Sag open reading frame. Either the recombinant 5019-5026. virus was generated in the HYB PRO/Cla transgenic females 11. Majors, J. & Varmus, H. E. (1983) Proc. Natl. Acad. Sci. USA 80, at levels below our detection and then selected in the non- 5866-5870. transgenic offspring or the recombination occurred in the 12. Brandt-Carlson, C., Butel, J. S. & Wheeler, D. (1993) Virology because were infected with copackaged trans- 185, 171-185. offspring they 13. Goodrich, D. W. & Duesberg, P. H. (1990) Proc. Natl. Acad. Sci. gene and Mtv-1 RNA. That we did see slow deletion of Vp14+ USA 87, 2052-2056. T cells in the HYB PRO/Cla mice indicates that some 14. Hu, W. & Temin, H. M. (1990) Science 250, 1227-1233. recombinatiori took place in these animals. In any case, if there 15. Stuhlmann, H. & Berg, P. (1992) J. Virol. 66, 2378-2388. were an alternative pathway of MMTV infection that did not 16. Coffin, J. M. (1990) in Virology, eds. Fields, B. N. & Knipe, D. M. require Sag activity, the parental HYB PRO/Cla virus retain- (Raven, New York), pp. 1437-1500. ing the frameshift mutation also should have been transmitted 17. Clavel, F., Hoggan, M. D., Willey, R. L., Strebel, K., Martin, to offspring, since it was the predominant species ofvirus shed M. A. & Repaske, R. (1989) J. Virol. 63, 1455-1459. into milk. Since only the repaired virus was found, it appears 18. Kozak, C., Peters, G., Pauley, R., Morris, V., Michalides, R., et that activity is required for viral transmission. at (1987) J. Virol. 61, 1651-1654. Sag 19. Morris, V. L., Medeiros, E., Ringold, G. M., Bishop, J. M. & Several groups have identified novel exogenous MMTVs Varmus, H. E. (1977) J. Mol. Biol. 114, 73-91. that encode Sag proteins of different V, specificities (23-25), 20. Callahan, R., Drohan, W., Gallahan, D., D'Hoostelaere, L. & and it was hypothesized that at least one of these may have Potter, M. (1982) Proc. Natl. Acad. Sci. USA 79, 4113-4117. arisen by recombination between an exogenous virus (SW21) 21. Golovkina, T. V., Tikhonenko, A. T., Vassetzky, N. S., III, Shef- and an endogenous virus (Mtv-6) (24). Moreover, sequence tel, B. I. & Gudkov, A. V. (1990) Virus Genes 4, 85-92. analysis of the various endogenous and exogenous MMTVs 22. Henrard, D. & Ross, S. R. (1988) J. Virol. 62, 3046-3049. indicates that some of these viruses were created by recom- 23. Held, W., Shakhov, A. N., Waanders, G., Scarpellino, L., Luethy, bination (12). We also proposed that copackaging of heterol- R., Kraehenbuhl, J.-P., MacDonald, H. R. & Acha-Orbea, H. RNAs and recombination might broaden the host range (1992) J. Exp. Med. 175, 1623-1633. ogous 24. Shakhov, A. N., Wang, H., Acha-Orbea, H., Pauley, R. J. & Wei, of MMTV (10). The V3 repertoire of outbred mice is deter- W.-Z. (1993) Eur. J. Immunol. 23, 2765-2769. mined in part by the endogenous Mtv proviruses present in 25. Yoshimoto, T., Nagase, H., Nakano, H., Matsuzawa, A. & their genomes (26), and exogenous virus can infect only those Nariuchi, H. (1994) Eur. J. Immunol. 24, 1612-1619. animals which do not have endogenous counterparts with the 26. Tomonari, K., Fairchild, S. & Rosenwasser, 0. A. (1993) Immu- same Sag specificity (4,5). The endogenousMtv proviruses will nol. Rev. 131, 131-168. Downloaded by guest on September 26, 2021