C3H Mouse Mammary Tumor Virus Superantigen Function Requires ...

JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

Oct. 2000, p. 9431–9440 Vol. 74, No. 20

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

C3H Mouse Mammary Tumor Virus Superantigen FunctionRequires a Splice Donor Site in the Envelope Gene

FARAH MUSTAFA, MARY LOZANO, AND JAQUELIN P. DUDLEY*

Section of Molecular Genetics and Microbiology and Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, Texas 78705

Received 10 May 2000/Accepted 25 July 2000

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is required for efficient milk-borne transmission of virus from mothers to offspring. The mRNA used for Sag expression is controversial, andat least four different promoters (two in the long terminal repeat and two in the envelope gene) for sag mRNAhave been reported. To determine which RNA is responsible for Sag function during milk-borne MMTVtransmission, we mutated a splice donor site unique to a spliced sag RNA from the 5* envelope promoter. Thesplice donor mutation in an infectious provirus was transfected into XC cells and injected into BALB/c mice.Mice injected with wild-type provirus showed Sag activity by the deletion of Sag-specific T cells and inductionof mammary tumors in 100% of injected animals. However, mice injected with the splice donor mutant gavesporadic and delayed T-cell deletion and a low percentage of mammary tumors with a long latency, suggestingthat the resulting tumors were due to the generation of recombinants with endogenous MMTVs. Third-litteroffspring of mice injected with wild-type provirus showed Sag-specific T-cell deletion and developed mammarytumors with kinetics similar to those for mice infected by nursing on MMTV-infected mothers, whereas thethird-litter offspring of the splice donor mutant-injected mice did not. One of the fifth-litter progeny of splicedonor mutant-injected mice showed C3H Sag activity and had recombinants that repaired the splice donormutation, thus confirming the necessity for the splice donor site for Sag function. These experiments are thefirst to show that the spliced sag mRNA from the 5* envelope promoter is required for efficient milk-bornetransmission of C3H MMTV.

Mouse mammary tumor virus (MMTV) is transmitted fromthe milk of infected mothers to the gut of susceptible offspring(33). MMTV infects B cells in the guts of newborn mice, andthese cells express the virally encoded superantigen (Sag) atthe plasma membrane in association with the major histocom-patibility complex (MHC) class II protein (1, 28). Sag is a typeII transmembrane protein that is required for efficient trans-mission of milk-borne MMTV from the gut to the mammarygland (12, 17, 23). The Sag-MHC complex is recognized byentire classes of T cells bearing particular T-cell receptor(TCR) b chains (18, 28). Recognition of Sag by specific T cellsleads to cell proliferation and/or release of cytokines, and thereleased cytokines recruit additional B and T lymphocytes thatare infected by MMTV (27). B-cell-deficient mice or micelacking Sag-reactive T cells cannot be efficiently infected byMMTV (4, 12). Ultimately, viral infection of the mammarygland is necessary to allow MMTV release into the milk. Micethat lack B cells or Sag-specific T cells also are defective in thespread of MMTV within the mammary gland (14). Thus, Sag isrequired for generation of a reservoir of virally infected B andT lymphocytes that are involved in MMTV transmission andviral spread to the mammary tissues.

The regulation of MMTV sag expression is controversial.Early studies indicated that Sag is translated from a singlyspliced mRNA that initiates at the U3/R border of the virallong terminal repeat (LTR) from the predominant U3 pro-moter (21, 41). This U3 promoter also drives expression of theviral structural genes, gag, pol, and env (for a review, see ref-erence 10) (Fig. 1). Cloning and sequencing revealed that this

sag mRNA uses a single splice donor site in the leader regionthat also is used for the generation of spliced envelope mRNA(32). The splice acceptor site for this sag mRNA is located inthe envelope region just upstream of the 39 LTR (21). Multiplestart codons are located near the 59 end of this mRNA, andmutagenesis experiments have suggested that the first or sec-ond codons can suffice for functional Sag production in cellculture (8). Subsequently, at least three other potential sagmRNAs have been described (Fig. 1). One of these mRNAsuses the same splice donor and acceptor sites as those de-scribed earlier, except that this transcript initiates approxi-mately 500 bp upstream of the standard viral RNAs (16).Another transcript initially was identified as a phorbol ester-inducible, cyclosporine-suppressible RNA in a T-cell lym-phoma (11, 31, 38). This sag-specific RNA is initiated from anintragenic promoter within the envelope-coding region; thetranscript uses a unique splice donor site within the enveloperegion and the same splice acceptor site as the other two sagRNAs. Most recently, a different sag promoter has been de-scribed within the envelope region (2). This promoter wasshown to be active in transient transfection experiments withreporter gene constructs. The resulting sag mRNA appears tobe unspliced and initiated within 100 bp of the 39 LTR (2).

Using PCR assays, our experiments have shown that all ofthe spliced sag transcripts are detectable in lymphocytes ofBALB/c mice that contain three endogenous MMTVs, Mtv-6,Mtv-8, and Mtv-9 (9, 24). However, only the spliced sag tran-script from the envelope promoter was detectable in cells in-fected in vitro or in vivo with milk-borne C3H MMTV. Simi-larly, deletion mutants of a C3H MMTV-derived infectiousmolecular clone (39) suggested that an intragenic envelopepromoter and an enhancer in the pol gene were responsible forsag expression (34, 35). To test whether the spliced mRNAfrom the env promoter is required for C3H MMTV sag expres-

* Corresponding author. Mailing address: Section of Molecular Ge-netics and Microbiology, The University of Texas at Austin, 100 W.24th St., Austin, TX 78705. Phone: (512) 471-8415. Fax: (512) 471-7088. E-mail: [email protected].

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sion, we used the C3H-derived infectious molecular clone toconstruct a mutant with alterations in the splice donor sitewithin the envelope region that is unique to this transcript.Stable transfections of this mutant or a control frameshiftmutant with an alteration within the sag coding region that hasbeen shown to abolish Sag function (13) produced virus-ex-pressing cell lines. Both the splice donor and frameshift mu-tants induced some mammary tumors with long latency andsporadic T-cell deletion after direct injection of transfectedcells. However, neither the frameshift nor the splice donormutant was transmissible to susceptible third-litter progenythrough the milk-borne route. These experiments show thatthe spliced mRNA from the envelope region is necessary andsufficient for efficient C3H MMTV milk-borne transmission.

MATERIALS AND METHODS

Mice. BALB/cJ mice were purchased from Jackson Laboratories (Bar Harbor,Maine). All animals were bred and maintained at the University of Texas atAustin Animal Resources Center. The animals were tested at periodic intervalsand were free of common bacterial and viral pathogens, including mouse hepa-titis virus. Four- to 5-week-old weanlings were injected with a total of 2 3 107 XCcells expressing MMTV proviral constructs and divided among five sites, foursubcutaneous injections near the mammary glands proximal to each leg and oneintraperitoneal injection as described by Shackleford and Varmus (39). All in-jected females were bred continuously to stimulate lactogenic hormones andMMTV production. Animals were palpated weekly for the appearance of mam-mary tumors.

Plasmid construction. Construction of the sag frameshift mutation at the ClaIsite in the 39 LTR of the HYB MTV provirus has been described previously (13).The splice donor sequence in the C3H MMTV envelope gene (nucleotide [nt]7339) was mutated using a PCR-based method. The choice of mutations wasbased on the conserved sequences encompassing the splice donor site. Since thesplice donor overlaps the coding sequences in the env gene, only the thirdposition in each codon was changed, except in one case, where a conservativevaline-to-leucine change was made (Fig. 1). The method for mutagenesis of thesplice donor site was essentially that described by Hoguchi (19). The first PCRwas performed using the sense oligonucleotide C3Hpol6361(1) (59 ATC TCACGT CAC GGG GAT CCC TTA CAA TCC 39) and the mutant antisenseoligonucleotide C3HenvSD7352(2) (59 GGA GAA AAt gag Agt CCc TGGTCA GGG AAG GCG CAA GGC AAC 39) (with mutant sequences lowercasedand boldfaced). (The primer numbering system corresponds to that for thecomplete BR6 provirus [32]). The second PCR was performed using the mutantsense oligonucleotide C3HenvSD7326(1) (59 CCT GAC CAg GGa cTc tca TTTTCT CCA AAA GGG GCC CTT GGG 39) and the antisense oligonucleotideC3Henv7519(2) (59 CTC TAT CAT TGG GAT CCT TAG GAG AAT TTTCCC 39). The final 1.3-kb product was gel purified, digested with BamHI, andligated to a 15-kb BamHI fragment from pHYB MTV, an infectious molecularclone of MMTV (39), to generate pHYB SD. The sequence of the clone wasconfirmed by automated fluorescent DNA sequencing.

Transfections. Stable cell lines of rat XC fibroblasts were generated by using10 ml of DMRIE-C (GIBCO BRL, Gaithersburg, Md.), 5 mg of wild-type ormutant CsCl-purified plasmid DNA, and 0.05 mg of DNA expressing the hygro-mycin expression cassette pTR174 (36). Transfections were performed in tripli-cate using six-well plates, and the cells were selected in Dulbecco’s modifiedEagle’s medium containing 7.5% fetal bovine serum (HyClone Laboratories, Inc.Logan, Utah), 50 mg of streptomycin/ml, 100 U of penicillin/ml, 200 mM glu-tamine, and 0.5 mg of hygromycin (GIBCO BRL)/ml until discrete colonies wereestablished. The colonies in the three wells were pooled and expanded. Thepooled clones were induced for MMTV expression using 1026 M dexamethasone(DEX) (Sigma Chemical Laboratories, St. Louis, Mo.), and a portion of thepooled population was used to make RNA, DNA, and proteins to assess MMTVexpression.

Immunoprecipitation and Western blot analysis. Whole-cell protein extractswere prepared from DEX-induced XC stable cell lines essentially as describedpreviously (26). The precleared lysates were incubated overnight with 1 ml ofanti-p27gag MMTV polyclonal antiserum (National Cancer Institutes/BiologicalCarcinogenesis Branch [NCI/BCB] Repository, National Institutes of Health) at4°C. The immune complexes were precipitated using 40 ml of anti-goat immu-noglobulin G (IgG)-agarose (Sigma) and separated on SDS-polyacrylamide gels(10 to 12% polyacrylamide). Separated immune complexes were transferred toOptitran nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) over-night at 4°C, blocked with 10% dried milk in a high-salt phosphate-bufferedsaline solution (0.2 M NaCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, and 2.6 mMKCl) containing 0.1% Tween 20, and incubated with anti-MMTV goat polyclonalantiserum (1:500 dilution) or monoclonal murine anti-SU (1:10 dilution ofBlue5; kindly provided by T. Golovkina, Jackson Labs). MMTV-specific proteinswere detected by using biotinylated anti-goat serum (Sigma) and horseradish

peroxidase-conjugated streptavidin (Calbiochem, Cambridge, Mass.) or horse-radish peroxidase-conjugated anti-mouse serum (Amersham Pharmacia BiotechLimited, Little Chalfont, United Kingdom). Proteins were visualized using theenhanced chemiluminescence kit as described by the manufacturer (Amersham).

Slot blot analysis. RNA was extracted from cultured cells or mouse tissuesusing the TRI Reagent as recommended by the manufacturer (Molecular Re-search Center, Cincinnati, Ohio). Dilutions of RNA were made and denatured ina slot blot RNA denaturation cocktail (0.693 SSC [13 SSC is 0.15 M NaCl plus0.015 M sodium citrate], 8.9% formaldehyde, and 69% formamide) and blottedonto a Zeta-Probe nylon membrane (Bio-Rad) using the Bio-Dot SF microfil-tration apparatus from Bio-Rad. The blotted filter was rinsed in 23 SSC and UVcross-linked twice at 1,200 mJ using a Stratalinker (Stratagene). The filter washybridized to a 1.4-kb probe detecting all MMTV mRNAs (a PstI fragmentfrom the 39 C3H LTR), while the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) probe was a PCR fragment containing sequences between nt 427 and983 of the murine GAPDH gene (37, 43). Probes had specific activities of ca. 108

cpm/mg of DNA. The blots were washed and subjected to autoradiography.RT-PCR. Twenty to forty micrograms of total cellular or tissue RNA was

DNase treated using 3 to 5 U of amplification grade DNase I (GIBCO BRL) at37°C for 1 h. DNase I was heat inactivated after the addition of EDTA to a finalconcentration of 2.5 mM and incubation at 70°C for 10 min. Five to ten micro-grams of DNase-treated RNA was used in the reverse transcription (RT) reac-tion with poly(dT17) primer. The primer was mixed with the RNA and boiled for5 min, followed by quick cooling on ice for 5 min. The denatured RNA wasreverse transcribed in a 50-ml reaction volume using 2 ml of Moloney murineleukemia virus (M-MLV) reverse transcriptase (GIBCO BRL) for 1 h at 37°C.Five microliters of cDNA was used in reactions containing 45 ml of SuperMix(GIBCO BRL) and 100 ng (;15 pmol) of each of the appropriate primers.Primer C3H LTR 420(2), 59 GAT TCA TTT CTT AAC ATA GTA AC 39, wasdesigned to specifically discriminate C3H-specific sequences from the endoge-nous MMTVs, Mtv-6, Mtv-8, and Mtv-9. The C3H LTR 420(2) primer was usedin combination with various sense primers to amplify (i) all MMTV-specificmRNAs (1551, 59 GGC ATA GCT CTG CTT TGC 39), (ii) sag mRNAs fromthe U3 promoters (2301, 59 GTG AAT TCC ATC ACA AGA GCG GAA CGGAC 39) (43), and (iii) sag mRNA from the 59 intragenic env promoter (72551, 59ATC GCC TTT AAG AAG GAC GCC TTC TTC T 39). Twenty microliters ofeach PCR product was analyzed by electrophoresis on 2% agarose gels andstained with ethidium bromide prior to photography.

Antibodies and flow cytometry analysis. Injected mice and their progeny werebled from the retro-orbital sinus at appropriate intervals. Peripheral blood lym-phocytes were purified using Histopaque (Sigma) and subjected to dual stainingfor CD4 and Vb14 as described by Wrona et al. (42). Antibodies were obtainedfrom PharMingen (San Diego, Calif.).

RESULTS

Construction of a splice donor mutant. At least four differ-ent RNAs have been described for the production of MMTVSag protein (2, 11, 16, 41) (Fig. 1A). Our previous work indi-cated that the spliced RNA initiated within the envelope re-gion was the only C3H sag-specific RNA detectable in cellsinfected by C3H MMTV (44). However, Sag is expressed inextremely small amounts (29), and it is possible that smallamounts of sag mRNA from alternative promoters would besufficient to allow Sag function in vivo. To determine if thespliced RNA that initiated in the envelope region was solelyresponsible for C3H MMTV Sag expression, we prepared amutant of the infectious MMTV clone, HYB MTV (39). Thismutant disrupted the splice donor site that is unique to thespliced sag mRNA from the envelope promoter (Fig. 1B). Ifthe spliced RNA from the envelope region is necessary for Sagproduction from C3H MMTV, mutation of this splice donorsite should eliminate C3H MMTV Sag function as measuredby deletion of reactive T cells and milk-borne transmission ofMMTV.

Because the splice donor site for C3H sag-specific RNA alsois located within the envelope-coding region, elimination of thesplice donor required a change of a single amino acid withinthe C3H envelope gene. To determine whether the proviruscontaining the splice donor mutation (HYB SD) could pro-duce sag-specific RNAs and MMTV proteins, we transfectedrat XC cells (which lack endogenous MMTVs) with the wild-type infectious clone or the proviral clone containing the splicedonor mutation. As a further control, we also transfected XC

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cells with an MMTV provirus containing a sag frameshift mu-tation, HYB CFS (Fig. 1A). The frameshift mutation would beexpected to truncate approximately the C-terminal two-thirdsof the Sag protein and eliminate Sag function (6, 13).

Total RNA was extracted from a pool of wild-type transfec-tants, three different pools of HYB SD transfectants (desig-nated SDI, SDII, and SDIII), or untransfected cells and ana-lyzed by RT-PCR for the production of sag RNA (Fig. 2).Using primers specific for the spliced sag RNA from the en-velope promoter, we detected this sag RNA in cells transfectedwith the infectious HYB MTV provirus, but not in pools ofcells transfected with HYB SD or untransfected controls (Fig.2A, lanes 2 and 3). Using conditions optimized from thosepublished previously (44), we also detected sag mRNA expres-sion from the U3 promoters in the LTR (Fig. 2B). Transfec-tants produced similar levels of total MMTV RNA, as mea-sured by RT-PCR and primers specific for the U3 region of theLTR, indicating that the failure to detect sag RNA in the splicedonor transfectants was not due to a general defect in viralRNA transcription (Fig. 2C). The general integrity of the RNAwas verified using RT-PCR and primers for GAPDH (Fig.2D). We independently verified that the sag frameshift muta-tion produced spliced sag mRNAs from the LTR and envelopepromoters (data not shown). These results indicated that theengineered mutation in the splice donor site eliminated pro-duction of the spliced sag RNA from the envelope promoter,but not from U3 promoters in the LTR.

Several other control experiments were performed to verifythe production of MMTV RNA and proteins from the HYBSD-transfected cells. The relative levels of total viral RNAwere determined in wild-type and mutant transfectants by im-mobilization of serially diluted RNA on nylon membranes andhybridization to an MMTV LTR probe (Fig. 3A). Levels of vi-ral RNA in the transfectants were quantitated by phosphor-

imager analysis after normalization to the level of GAPDHRNA in each sample (Fig. 3B). These results showed that thelevel of total viral RNA in splice mutant or frameshift mutanttransfectants was approximately the same as that in wild-typetransfected cells. MMTV Gag protein production in wild-typeand mutant transfectants also was determined by Western blot-

FIG. 1. Diagram of the MMTV sag-specific mRNAs and the location of the splice donor mutation. (A) Schematic representation of the MMTV genome andreported MMTV sag mRNAs. The boxes on the MMTV proviral genome show the positions of the indicated open reading frames and the LTRs. The reported sagtranscripts are indicated below the provirus; introns are indicated by dotted lines, and exons are represented by dashed lines with arrows. The figure also shows thelocations of the reported MMTV promoters and splice sites used for sag gene expression, as well as primers used to differentiate among sag mRNAs. SD, splice donor;SA, splice acceptor; CFS, ClaI frameshift. (B) Comparison of the mutant splice donor (mSD) site in env with the sequence of wild-type (WT) C3H MMTV. The boxedarea highlights the mutated region of the splice donor. The underlined letters reflect the canonical GT of the splice donor, while the bold amino acids reflect theconservative change made by the mutation.

FIG. 2. The splice donor mutation abrogates expression of sag mRNA fromthe 59 intragenic env promoter. RT-PCR analysis was performed using RNAextracted from XC cells transfected with the wild-type or SD mutant proviruses.(A) sag mRNAs expressed from the 59 env promoter; (B) sag mRNAs expressedfrom U3 promoters; (C) all MMTV mRNAs; (D) GAPDH mRNA as a controlfor RNA and cDNA integrity. Lane 1, XC control RNA from untransfected XCcells; lane 2, RNA extracted from a pool of wild-type HYB MTV transfectants;lane 3, RNA extracted from a pool of HYB SD transfectants.

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ting (Fig. 4A). Transfected cell lysates were immunoprecipi-tated with MMTV CA-specific antisera followed by Westernblotting with anti-MMTV sera. Although this technique is notas quantitative as the slot blot assays, very similar levels ofMMTV protein expression were observed in HYB MTV- andHYB SD-transfected cells (Fig. 4A). Western blots of trans-fected cell extracts (without prior immunoprecipitation) usingMMTV SU-specific antisera revealed that the splice donor mu-tant was capable of producing SU protein (Fig. 4B). These ex-periments suggested that the splice donor mutation in theenvelope region did not dramatically affect total MMTV RNAlevels or MMTV Gag or Env protein production. Efforts to quan-titate MMTV production from XC cells have been hamperedby low levels of virion release (F. Mustafa, unpublished data).

To determine whether the SD mutation in the envelopegene altered the overall splicing pattern in MMTV-transfected

cells, RNA was extracted from wild-type- or mutant-trans-fected XC cells and subjected to Northern blotting. Althoughlevels of sag mRNA in most cell types are insufficient fordetection on Northern blots, no reproducible differences in theratio of gag-pol to env mRNAs were observed between wild-type and mutant transfected cells (data not shown).

In vivo infection with sag mutants. Our data indicated that asplice donor mutation in the envelope region eliminated theability of an infectious MMTV provirus to produce the splicedsag-specific RNA from the envelope promoter. However, thissame mutation did not eliminate the ability of the virus toproduce MMTV RNA and Gag or Env proteins. Therefore,wild-type HYB MTV transfectants, sag frameshift transfec-tants, and three different pools of HYB SD transfectants wereassessed for the ability to transmit MMTV in vivo after injec-tion into weanling BALB/c mice.

Deletion of Sag-reactive T cells is a sensitive indicator ofMMTV infection as well as Sag function (28). Thus, injectedmice were tested for the deletion of CD41 Vb141 T cellsreactive with C3H MMTV Sag. As expected, mice injectedwith the HYB MTV-transfected XC cells showed approxi-mately 30% deletion of Vb141 T cells within 2.5 months ofinjection compared to uninjected mice, whereas pools of HYBSD-transfected cells did not (Fig. 5). Mice also were testedat 4 months postinjection. The HYB MTV-injected animalsshowed 50% deletion of Sag-cognate T cells, whereas no de-tectable deletion was apparent in any of the mice injected withHYB SD transfectants. However, at approximately 8 monthspostinjection, we observed deletion of Vb141 T cells in 2 of 16animals (12.5%) injected with the SD transfectants and 1 of 6animals (17%) injected with the sag frameshift mutant. Fur-thermore, deletion of Sag-cognate T cells was not observed inthe surviving mice 14 months after injection with HYB SD-transfected cells. These results suggested that the mutation inthe HYB SD provirus that prevented production of the splicedsag-specific RNA from the envelope promoter also interferedwith Sag function, as demonstrated by the ability to deleteSag-specific T cells.

Injected animals also were monitored for MMTV infectionby the appearance of MMTV-induced mammary tumors. All ofthe HYB MTV-injected female mice (4 of 4) developed mam-mary tumors between 6 and 8 months of age (average latency,7 months) (Table 1). In contrast, 5 of 12 females (42%) in-jected with SD transfectants developed mammary tumors, with

FIG. 3. Total MMTV RNA expression from different XC cell transfectants isequivalent. RNAs extracted from pools of transfected XC cells were diluted andsubjected to slot blot analysis. Blots were hybridized to 32P-labeled probes for theMMTV LTR (A) or GAPDH (B). All RNAs were treated with DNase I prior toblotting. Relative expression of MMTV RNAs in mutant-transfected cells wascalculated by using values that were obtained by phosphorimager analysis usinghybridization of 0.2 and 2.0 mg of RNA to the LTR probe and then normalizedfor RNA loading by using the values obtained with the GAPDH probe.

FIG. 4. HYB MTV- and mutant-transfected cell lines express MMTV-specific Gag and Env proteins. (A) Expression of MMTV Gag proteins using immunopre-cipitation and Western blot analysis. A polyclonal antibody against MMTV CA protein was used for immunoprecipitation, followed by detection with anti-MMTVpolyclonal antibody. There is cross-reactivity of the antibodies directed against MMTV-specific proteins with XC cell proteins. Some minor bands were observedinconsistently in different extracts, presumably due to some protein degradation or processing, but major MMTV protein levels were similar in mutant and wild-typetransfectants. The positions of MMTV-specific proteins and precursors are shown on the right, and molecular weight markers are shown on the left. (B) Expressionof MMTV Env protein using Western blot analysis with a monoclonal gp52 (SU)-specific antibody.



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a latency of 9 to 15 months (average latency, 12 months).However, only one of the four tumor-bearing mice testedshowed 40% deletion of Vb141 T cells, and this animal alsodeveloped a mammary tumor, suggesting that mammary glandinfection in these animals may have occurred due to genera-tion of recombinants with endogenous MMTVs (see Discus-sion). One of four females injected with XC cells expressingthe sag frameshift mutant also developed a mammary tumor;this mouse showed 25% deletion of Vb141 T cells at approx-imately 1 year postinfection (Table 1).

RNA was obtained from several tissues of animals injectedwith HYB MTV or HYB SD transfectants and was used forRT-PCR with C3H MMTV-specific primers within the LTR(Fig. 6A, panel I). Five different HYB SD-injected animalsshowed MMTV infection of multiple tissues, including themammary gland, spleen, lymph nodes, and salivary glands(lanes 5 to 11; data shown for two animals only). To determinewhether the RNA detected in mammary glands was due toreversion of the injected HYB SD virus at the splice donor site,we used RT-PCR and primers within the envelope gene thatwere specific for the splice donor mutation (Fig. 6A, panel II).These assays showed that RNA from the splice donor mutant

was expressed in each of these tissues, confirming that a gen-eralized infection of the mice had occurred (lanes 5 to 11).Such RNAs were not detectable in mammary tumors inducedby injections with the wild-type HYB MTV (Fig. 6A, panel II,lanes 3 and 4) or in uninjected BALB/c mice (lanes 1 and 2).Additional PCRs revealed that the spliced sag-specific RNAfrom the envelope promoter was not detectable in RNA ex-tracted from mammary tumors of HYB SD-injected mice (Fig.6B, lanes 3 and 4); however, this RNA was detectable in RNAsextracted from mammary tumors of HYB MTV-injected ani-mals (Fig. 6B, lanes 1 and 2). These results suggested that thesplice donor mutant lacks Sag function, as demonstrated by thefailure to reproducibly delete Sag-reactive T cells, but that themutant or its recombinants are capable of infecting the mam-mary gland and other tissues following injection of infected XCcells.

Lack of milk-borne transmission by sag mutants. BecauseSag function appears to be required for efficient transmissionof milk-borne MMTV (13), we tested the progeny of HYBMTV-, HYB SD-, and sag frameshift mutant-injected animalsfor evidence of MMTV infection. As anticipated, offspring ofHYB MTV-injected mice showed approximately 20% deletion

FIG. 5. Sporadic deletion of Vb141 CD41 T cells in mutant-injected BALB/c mice. Deletion of cognate T cells determined by fluorescence-activated cell sorter(FACS) analysis is shown at various times after injection of XC cell transfectants. Each time point represents FACS analysis of the peripheral blood lymphocytes fromone to three mice. Standard deviations from the means are indicated for each time point measured. Heavy arrows indicate the average latency of mammary tumorsinduced by the wild-type or mutant viruses.

TABLE 1. Vb141 T-cell deletion and mammary tumor development in injected mice

Construct No. of F/Ma

mice injected

No. of mice withdeletions of Vb141 Tcells/total no. of mice

No. of mice withmammary tumors/

total no. of females

Mammary tumorlatency period

(mos)

Avg tumor latencyperiod (mos)

HYB MTV 4/2 6/6 4/4 6–8 7HYB SD 12/4 2/16b 5/12c 9–15 12HYB CFS 4/2 1/6 1/4 13 13

a F, female; M, male.b Only one of the two mice with deletions of Vb141 T cells developed a mammary tumor.c One of the mammary tumor-bearing mice showed Vb141 T-cell deletion, whereas three did not. The remaining tumor-bearing mouse was not tested.



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of C3H Sag-specific T cells at 2.5 months of age (Fig. 7), andthis deletion increased with age. Furthermore, offspring ofHYB MTV-injected animals (three of three females) devel-oped mammary tumors with an average latency of 9.5 months(Table 2). In contrast, third-litter progeny mice injected withHYB SD or sag frameshift mutants did not delete CD41

Vb141 T cells at any age tested (up to 1 year). No mammarytumors have developed in the offspring of animals injected withHYB SD mutants (0 of 5 females; Table 2). Together theseresults indicate that elimination of the spliced sag-specificRNA from the envelope promoter is sufficient to abolish C3HMMTV Sag function.

Appearance of MMTV recombinants that regenerate thesplice donor site in the envelope gene. Because we observedsporadic deletion of Sag-reactive T cells and long-latency tu-mors in injected mice, we suspected that recombinants be-tween the injected mutant viruses and endogenous MMTVswere being generated to correct the defective sag genes. There-fore, we used RNA extracted from injected mice or their third-litter progeny to perform RT-PCR with primers that wouldamplify the sag RNA splice junction and simultaneously detectsequences specific for the C3H MMTV LTR. The resultingproduct spanned a ClaI site in the C3H MMTV U3 region(Fig. 8A). Because the endogenous MMTVs of BALB/c mice(Mtv-6, -8, and -9) lack a ClaI site at this position, failure tocompletely digest the PCR product would indicate the pres-ence of recombinants. As expected, ClaI digestion of PCRproducts from mammary tumors of HYB MTV-injected mice

or their third-litter progeny showed complete digestion prod-ucts of 487 and 111 bp, consistent with the presence of splicedsag RNA from the envelope promoter (Fig. 8B, lanes 2, 3, and11). A mammary tumor from a mouse injected with the ClaIframeshift mutant expressed the spliced sag mRNA, but theRT-PCR product was not digested with ClaI, as anticipated(lane 9). RNA from mammary tumors of the HYB SD-injectedmice gave the product from the gag/pol and env mRNAs, butnot the product from the spliced sag mRNA (lanes 4, 5, 6, and7). The mammary tumor shown in lane 8 also expresses re-combinant MMTVs, but these recombinants are not detectableby the C3H-specific primer used (data not shown). Third-litteroffspring of HYB SD-injected mice had no C3H-specific prod-ucts detectable in salivary gland RNA (the mammary glandwas not available) (lanes 12 to 14), but RNA from the mam-mary gland (or salivary gland) of a fifth-litter female thatshowed deletion of C3H Sag-specific T cells allowed detectionof an RT-PCR product of the size expected for sag mRNA.However, most of the product was not digested with ClaI,although a control plasmid in the same reaction was digestedcompletely (lanes 17 and 18). Sequencing analysis confirmedthat this PCR product was derived from a recombination be-tween C3H MMTV and Mtv-9 that regenerated a functionalsplice donor site in the envelope region, while retaining C3Hsequences in the Sag region controlling TCR interaction (Fig.8C). The ClaI-digested products probably result from a differ-ent recombinant that repaired the splice donor mutation butretains a functional ClaI site in the LTR. Together with pre-

FIG. 6. Spread of MMTV infection in mice injected with XC cells expressing the splice donor mutant virus. (A) Detection of MMTV-specific RNA in the variousorgans of injected mice using RT-PCR. (Panel I) RT-PCR with primers specific for C3H MMTV LTR sequences; (panel II) RT-PCR with primers specific for the splicedonor mutation; (panel III) RT-PCR specific for GAPDH RNA. Tissues analyzed: LN, lymph node; SP, spleen; MG, mammary gland; MT, mammary tumor; SG,salivary gland. In some cases, results from two individual mice are shown. Spontaneous mammary tumors in our BALB/c colony are rare (,1%). (B) Expression ofsag mRNAs from the intragenic env promoter in the mammary tumors from wild-type (HYB MTV) (lanes 1 and 2)- and SD mutant (lanes 3 and 4)-injected mice.Results from two individual mice are shown. Because a large number of cycles was used for PCR assays to detect sag mRNA, these results are not quantitative. Inaddition, use of the primer pair for the splice donor mutation appears to be more sensitive for PCR assays than the C3H LTR primer pair used in panel A. Expressionof GAPDH was monitored by RT-PCR as a control for RNA and cDNA integrity. RT-PCR products were separated on 2% agarose gels and visualized by ethidiumbromide staining prior to photography.



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vious experiments, these results suggest that recombinants gen-erated in injected mice are selected during milk-borne trans-mission for regeneration of the splice donor site in theenvelope gene.

DISCUSSION

Requirement for the splice donor site in the envelope genefor C3H Sag function. Previous experiments indicate that Sagis required for efficient milk-borne MMTV expression as wellas for viral spread within the mammary gland (12, 13, 17).Because four different MMTV promoters have been impli-cated in sag mRNA expression (2, 11, 16, 41), we have mutatedthe splice donor site unique to the spliced sag-specific mRNAfrom the intragenic envelope promoter in the context of aninfectious MMTV provirus (see Fig. 1). Injection of HYBSD-transfected XC cells into weanling BALB/c mice revealeddelayed and sporadic MMTV infection as assessed by deletionof Sag-cognate T cells, infection of mammary glands and othertissues, and the appearance of mammary tumors compared tofindings for mice injected with wild type-transfected cells. Thediscrepancies between the wild-type and mutant infectionswere not due to differences in overall MMTV RNA productionor splicing patterns in the original injected XC cells, but were

correlated specifically with the absence of the spliced sagmRNA from the 59 intragenic envelope promoter.

Although some animals could be infected with the HYB SDvirus by direct injection, the splice donor mutant was not trans-mitted to newborn BALB/c progeny by the normal milk-borneroute. Specifically, third-litter progeny of HYB SD-injectedmice lacked deletion of Sag-cognate T cells and failed to de-velop mammary tumors. Such results are identical to thoseobtained by Wrona et al. (42) using HYB MTV containingsubstitution mutations within the carboxyl-terminal Sag resi-dues. The Sag C-terminal amino acids at the surface of anti-gen-presenting cells are required for interactions with the TCR(45). Experiments by Pullen and colleagues (30) also haveshown that most amino acid substitutions in the Sag C termi-nus are sufficient to abolish functional Sag expression. Theability of SD mutants to infect the mammary gland by directinjection, but not by the milk-borne route, might be explainedby the higher infectivity of cell-associated virus, by an alterna-tive infection pathway mediated by XC cells, or by sporadicappearance of recombinants (see below). However, the similareffects of mutations in the splice donor site within the envelopegene and at the ClaI site within the sag gene suggest that thesplice donor site in the envelope gene is necessary for C3H

FIG. 7. The splice donor mutant viruses are defective in milk-borne transmission of virus from mothers to offspring. The kinetics of Vb141 CD41 T-cell deletionis shown for the third litters of the injected mice. Each time point represents fluorescence-activated cell sorter analysis of the peripheral blood lymphocytes from twoto six mice. Standard deviations from the means are indicated. The average latency of mammary tumors induced by the wild-type HYB MTV-derived virus is shown.

TABLE 2. Vb141 T-cell deletion and mammary tumor development in the third litters of injected mice

Virus injected intothird litters

of mice

No. ofF/Ma

No. of mice withdeletions of Vb141 Tcells/total no. of mice

No. of mice withmammary tumors/

total no. of females

Mammary tumorlatency period

(mos)

Avg tumor latencyperiod (mos)

HYB MTV 3/4 7/7 3/3 9–10 9.5HYB SDb 5/8 0/13 0/5 NAc NAHYB CFSb 5/1 0/6 0/6 NA NA

a Number of females tested/number of males tested.b All animals were monitored for 1 year.c NA, not applicable.



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MMTV Sag expression and its function in the milk-borne routeof transmission.

If the spliced sag mRNA from the envelope promoter isrequired for Sag function, why do HYB SD-injected mice showsporadic deletion of Sag-reactive T cells and long-latencymammary tumors? Because similar results were obtained withmice injected with the sag frameshift mutant, we believe thatthese results are most readily explained by the generation ofrecombinants between the endogenous MMTVs of BALB/cmice and the splice donor mutants, rather than by the functionof another sag mRNA. Indeed, we have shown that such re-combinants are generated, and recombinants that repair thesplice donor defect are transmitted to the progeny of inject-ed animals (Fig. 8). Recombinants generated by the sag frame-shift virus were previously reported in C3H transgenic mice(13), and recombinants between endogenous and exogenousMMTVs also have been observed in the BALB/cT substrain ofmice (15). If such recombinants are generated, why do we notobserve deletion in third-litter progeny of SD-injected mice?We believe that efficient Sag function requires recovery of thesplice donor site through recombination. However, additionalrecombination events may be required to generate a virus witha wild-type splice donor site that also produces a wild-type(C3H-like) Sag reactive with Vb141 T cells. Sequence analysisof a recombinant observed in a fifth-litter progeny of HYBSD-injected mice suggested that multiple crossovers were nec-essary to generate such a recombinant. This litter was born tomothers 10 months postinjection, while the third litters wereborn to females 3 to 5 months postinjection (data not shown).The production of revertants of the splice donor mutation thatare selected during milk-borne MMTV transmission arguesstrongly that this sequence is necessary for Sag function. To-gether, our data indicate that the spliced sag mRNA from theenvelope promoter is the major functional sag mRNA pro-duced from the C3H MMTV provirus.

Expression of other sag mRNAs. In previous RT-PCR ex-periments, we were unable to detect spliced sag mRNAs fromthe C3H MMTV LTR in tissues infected by the virus (44).However, using more sensitive conditions, we have been ableto detect sag mRNAs from the LTR promoters in XC cellstransfected with the HYB MTV provirus (Fig. 2). Sequencingof these products revealed a single splice donor site identical tothat used for spliced env mRNA (11) (data not shown). Thesingly spliced sag mRNA from the LTR promoters also sharesa splice acceptor site with the spliced sag mRNA from theenvelope promoter (40, 41). These results indicate that the sagmRNAs from the LTR promoters contain functional splicedonor and acceptor sites. Clearly, functional sag mRNAs fromthe LTR promoter can be synthesized, since Mtv-6 encodes aSag protein that causes intrathymic deletion of CD41 Vb31

and Vb51 T cells (3, 7, 42). This Sag expression must originatefrom the LTR promoters because the Mtv-6 provirus has alarge internal deletion that includes most of the gag, pol, andenv sequences (7).

If sag mRNA is synthesized from the C3H LTR promoter(s),why is this RNA nonfunctional? The most obvious explanationis that sag mRNA expression from the LTR promoters is sup-pressed in antigen-presenting cells, whereas the envelope pro-moter that produces a singly spliced sag transcript is active inthese cells. Our laboratory has identified several negative reg-ulatory elements (NREs) that suppress expression from theC3H MMTV LTR promoter in the lymphoid tissues of trans-genic mice (5, 20). Transfection experiments by Miller et al.(31) suggested that the env promoter is most active in T-celllines, rather than B-cell lines, but this does not preclude Sagexpression in B cells or other antigen-presenting cells in vivo.Since the sag transcripts from the LTR and env promoters aredetected with different primer pairs, it is difficult to quantitatedifferences in the levels of these RNAs. Transcription of sagRNA from the LTR or envelope promoters also leads to dif-ferences in the 59 untranslated regions of these mRNAs. Suchdifferences may affect translation or transport of the mRNAsin antigen-presenting cells. Moreover, translation and trans-port inefficiencies of sag mRNAs from the LTR promoter(s) inB cells would be magnified by transcriptional suppression me-diated by the NREs in these cells. As pointed out by Wrona etal. (42), the use of separate promoters for sag mRNA and thestructural genes allows MMTV to optimize Sag expression, butnot virus production, in lymphoid cells. Separate promotersalso exist for the structural genes and accessory genes of thefoamy viruses (for a review, see reference 25). In these viruses,the transactivator Tas or Bel-1 has a higher affinity for theenvelope promoter than the LTR promoter, allowing a switchto structural gene transcription later in the infectious cycle(22).

Our experiments provide no evidence that the envelope pro-moter reported near the 39 end of the envelope gene (2) is usedto make a functional unspliced mRNA from the C3H MMTVprovirus (see Fig. 1A). Although we cannot rule out synthesisof this unspliced sag RNA from the C3H MMTV provirus, ourexperiments with the splice donor mutant in the envelope genesuggest that potential sag mRNAs from this 39 envelope pro-moter are not sufficient for Sag activity required for milk-borneMMTV transmission. Transient transfection experiments byReuss and Coffin (34) using deleted forms of the HYB MTVcarrying a reporter gene in the sag open reading frame alsosupport this conclusion. Thus, our data and those from othersindicate that functional Sag expression from C3H MMTV oc-curs from the intragenic envelope promoter that producesspliced sag mRNA.

FIG. 8. Recombinants from fifth-litter progeny of HYB SD-injected mice repair the splice donor mutation. (A) Diagram showing the primers used and positionsof ClaI cleavage sites in RT-PCR products from MMTV-infected mice. The sizes of digested and undigested products are given. (B) Cleavage of RT-PCR productsgenerated using a primer just upstream of the splice donor site in the envelope region [7255(1)] and a C3H-specific primer in the LTR [420(2)]. RNA from miceinoculated with XC transfectants of wild-type (HYB MTV) or mutant proviruses (HYB SD and HYB CFS) was used for RT-PCRs shown in lanes 2 to 9. Of the splicedonor mutant-injected mice, only the mouse in lane 4 showed C3H Sag-specific T-cell deletion. RNA from the progeny of mice inoculated with wild-type or mutanttransfectants was used in lanes 11 to 18. None of the third litters tested (total, 13 mice) showed any C3H Sag-specific T-cell deletion, whereas one fifth-litter femaleshowed .50% deletion. The undigested bands representing spliced sag mRNA from the envelope promoter in the lactating mammary gland of the fifth-litter female(lanes 17 and 18) were excised and subjected to sequencing. RT-PCR products were purified using Micro Bio-Spin P-30 chromatography columns (Bio-Rad) prior toincubation with ClaI in the presence of a plasmid digestion control (see arrows). The integrity of cDNA samples was assessed using GAPDH primers for PCRs (lowerpanel). MT, mammary tumor; CFS, ClaI frameshift virus; SG, salivary gland; SP, spleen; LMG, lactating mammary gland. (C) Comparison of sequences from therecombinant (REC), C3H MMTV, and endogenous Mtv-9. The sequence of the recombinant virus was obtained after isolation of the 598-bp band shown in lanes 17and 18 of panel B. Only the portion of the splice donor mutation that is present in the spliced sag RNA is shown. The positions of AvrII, ClaI, and C3H-specificoligonucleotide primers are shown. Note that the recombinant analyzed appears to be the result of multiple recombination events, since the REC sequence is Mtv-9-likearound the splice junction, C3H-like around the AvrII site, Mtv-9-like around the ClaI site, and C3H-like at the 39 end due to the primer used for PCR.



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ACKNOWLEDGMENTS

We thank Susan Ross and members of the Dudley laboratory foruseful comments on the manuscript. We also acknowledge the help ofAlexandra Mey in the construction of the splice donor mutant.

This work was supported by grants R01 CA34780 and CA52646from the National Institutes of Health. F.M. is a recipient of an NIHNRSA award.

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