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 Previous Article

Journal of Virology, January 2004, p. 1055-1062, Vol. 78, No. 2
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.2.1055-1062.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

RIII/Sa Mice with a High Incidence of Mammary Tumors Express Two Exogenous Strains and One Potential Endogenous Strain of Mouse Mammary Tumor Virus

Nurul H. Sarkar,1* Tatyana Golovkina,2 and Taher Uz-Zaman1,{dagger}

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912,1 The Jackson Laboratory, Bar Harbor, Maine 046092

Received 1 July 2003/ Accepted 28 September 2003


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ABSTRACT
 
The inbred mouse strain RIII has long been known for shedding large amounts of mouse mammary tumor virus (MMTV) particles in milk and for the development of hormone-dependent early mammary tumors at a very high incidence (>90%). We have established one RIII subline (RIII/Sa) that shows a pattern of virus expression and tumor incidence similar to that in RIII mice. In the present study, we analyzed the milk and mammary tumors of RIII/Sa mice for virus characterization by reverse transcriptase PCR (RT-PCR) cloning and sequencing of the open reading frame (ORF) of the MMTV long terminal repeats (LTRs). Our results show that these mice express a mixture of at least three different MMTV strains, two of which, designated here as RIII/Sa MMTV-1 and RIII/Sa MMTV-2, are exogenous. The third virus, RIII/Sa MMTV-3, appears to carry the signature of an endogenous provirus, Mtv-17. Similar studies done with the milk and mammary glands of another subline, RIIIS/J, revealed that they do not express MMTV in their milk. The RIII/Sa and RIIIS/J mice also exhibited differences in their endogenous proviral contents. Twelve spontaneously developed mammary tumors of RIII/Sa mice were examined for possible Wnt-1 and/or int-2/Fgf3 mutations that are usually found to occur in most mouse mammary tumors as a consequence of MMTV proviral integration. This work led to the isolation of one MMTV-Wnt-1 junction fragment and one MMTV-int-2/Fgf3 junction fragment from 2 of the 12 tumors. Further analyses showed that both junction fragments contained the RIII/Sa MMTV-2-specific LTR ORF, indicating that this virus was involved in the development of both tumors. Whether RIII/Sa MMTV-1 and/or RIII/Sa MMTV-3 plays any role in mammary tumor development in RIII/Sa mice remains to be established. Overall, the present study demonstrates, to our surprise, that (i) RIII/Sa mice express, unlike other native mouse strains, three strains of MMTVs; and (ii) the virions are completely different from the virus expressed by another subline of RIII mice, the BR6 mice.


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INTRODUCTION
 
Bittner observed in 1936 that the presence of an agent in the milk (milk factor) of an inbred laboratory strain of mice (C3H) closely correlated with its potential for mammary tumor development (2). This discovery led to countless investigations into the biology of mammary tumors in a number of standard laboratory strains of mice, particularly, C3H, RIII, A, and GR. Some work has also been done with BR6 mice, a strain that was derived by breeding a C57BL female mouse with an RIII male (10). Bittner's milk agent was later identified to be an exogenous mouse mammary tumor virus (MMTV) that is transmitted from viremic mothers to their offspring via milk and induces mammary tumors (6). However, during the early 1970s, some laboratory mouse strains were shown to carry variable copies of MMTV proviruses (endogenous proviruses) in their genome irrespective of whether they shed MMTV particles in their milk and/or develop mammary tumors (36). It is now known that all inbred strains of mice, as well as some wild mice, contain multiple copies of MMTV proviral integrants (Mtvs), although most of them fail to produce infectious MMTV particles. Interestingly, some Mtvs, such as Mtv-2 in strain GR and Mtv-1 in strain C3H, are expressed in mammary glands, leading to virus production and shedding in milk (23). However, these two viral strains differ in their biology: while the GR Mtv-2 virus induces mammary tumors at an early age (<6 to 8 months) and at a high incidence (>90%), tumors developed by the C3H Mtv-1 virus have a long latency period (>20 months), and their incidence is not high (<50%). More importantly, the virions that these mice produce are also transmitted as infectious agents (exogenous) when newborn mice suckle on viremic females. Other mouse strains, such as A and RIII—which have been assumed to produce early in life only one strain of milk-borne exogenous MMTV at high levels and to induce early mammary tumors at a high incidence (>90%)—are also known to express smaller amounts of some endogenous proviruses. For example, foster-nursing of newborn RIII pups to milk-borne-MMTV-free BALB/c or C57BL mice results in significantly lowering the incidence of mammary tumors (<10%) that arise late in life (>20 months). The inducer of such late-developing tumors in RIII/Saf mice has been assumed to be an endogenous MMTV, and the virus has been found to be shed in smaller amounts in the milk of only high-parity mothers (23).

Further studies on MMTV pathogenesis led to three other important observations. First, MMTV superantigen (Sag), a product of the open reading frame (ORF) contained within the 3' long terminal repeat (LTR) of the virus, was shown to interact with the Vß portion of specific T-cell receptors (TCrs) in MMTV-infected mice, causing virus infection and deletion or activation of certain T cells (3, 8, 11, 22, 40). These lymphoid cells serve as reservoirs for infection of the mammary gland. Second, MMTV does not carry any oncogene in its genome, and thus the mechanism by which it induces mammary tumors in mice is the ability of the virus to act as an insertional mutagen and activate the expression of an int family of cellular oncogenes (5, 12, 27). Finally, recombination between endogenous and exogenous viruses has been shown to result in the generation of new virus strains with a broader host range. For example, the endogenous Mtv-1 locus in C3H/HeN mice is highly transcribed in lactating mammary glands, but little or none of this viral RNA is packaged into virions. However, when these mice are infected with exogenous C3H MMTV, the endogenous Mtv-1 RNA is copackaged into virions, and the resultant recombinant virus infects mice with a broader host range than the parental C3H MMTV (13). Formation of new milk-borne MMTVs via recombination between endogenous and exogenous viruses has also been found in BALB/cT mice (14). Recombination between nonpathogenic (Mtv-17) and highly pathogenic (Mtv-2) endogenous MMTVs has also been observed to occur in GR mice (15). Since the presence of endogenous and/or exogenous MMTVs in different strains of mice is highly variable, it seems likely that specific recombination events between endogenous and exogenous viruses may lead to the expression of different MMTV strains in the milk of different mouse strains. In addition, some mouse strains may produce different strains of native proviruses. In fact, two different types of viruses with different biological characteristics have been shown to be present in at least one mouse strain, TES2 (26). These observations prompted us to address the question of whether or not low-parity RIII mice (designated henceforth as RIII/Sa) express multiple MMTV strains and, if so, to characterize the proviruses on the basis of their ORF sequences.


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RIII/Sa mice express three strains of MMTVs.
 
The progeny of a mammary tumor-bearing 7-month-old RIII mouse (4th parity) was isolated in 1974 and maintained as a separate lineage, designated RIII/Sa, in the laboratory of N. Sarkar. This subline has been maintained since 1974 by brother-sister mating of young adults from high-parity (3rd or more) mothers (30). RIII/Sa mice produce large quantities of MMTV in their milk (1011 to 1012 particles/ml) and develop early mammary tumors (within 6 to 10 months) at a very high incidence (>90%). To characterize the milk-borne MMTV expressed by low-parity RIII/Sa mice and early mammary tumors, we took advantage of the fact that sequences near the 3' end of the ORFs of different MMTV strains are polymorphic (41) and thus can be used to identify the source of the viral genome. MMTV particles were partially purified from the milk of RIII/Sa mothers of early and late parities (31). Virus was also isolated from the milk pellet obtained from the stomachs of 2- to 4-day-old pups (13). Viral RNA, as well as RNAs from mammary tumors, mammary glands, and spleens, was isolated with the TRIzol kit (GIBCO-BRL) according to the instructions provided by the supplier. To remove possible DNA contamination, RNA samples were subjected to poly(A) column purification with the Oligotex kit (QIAGEN). Each RNA sample was subjected to reverse transcriptase PCR (RT-PCR) with a pair of primers containing sequences common to most MMTV LTRs. The forward primer, FPC1, represented a region located about 400 bp upstream to the R domain of the LTR, while the reverse primer (RP) contained sequences from the 3' end of the U3 and R regions (Fig. 1A). The primers FPC1, FPC2, and RP were designed by using the published sequences of BR6 MMTV (25). Analyses by gel electrophoresis of the RT-PCR amplification products that were synthesized with viral RNA from 15 different milk samples and 12 mammary tumors using the primers FPC1 and RP (Fig. 1) showed the presence of products of two different sizes. The results obtained with two such milk and two tumor samples are shown in Fig. 1B (lanes 2 and 4 and 6 and 8, respectively). For brevity, we have designated the cDNAs on the basis of their source (milk, M; or tumor, T) and size (large, L; or small, S) as ML, MS, TL, and TS, respectively. It should noted that the relative amounts of the large and smaller PCR products obtained from milk samples appeared to be similar (lanes 2 and 4), whereas the smaller products produced by tumor samples were significantly smaller in amount than the larger products (lanes 6 and 8). Furthermore, 3 of the 12 tumors were negative for the TS cDNA, and the TS products varied in amount from one sample to the other. Overall, our observation indicated that RIII/Sa mice express in their milk and mammary tumors at least two different strains of MMTVs that differ in their LTR sequences. We have designated these viruses as RIII/Sa MMTV-1 (representing the MS and TS bands) and RIII/Sa MMTV-2 (representing the ML and TL bands).



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  		FIG. 1. (A) Schematic diagram showing a small segment of the env gene and the entire LTR domain of BR-6 MMTV (25). The approximate locations of the forward (FPC1, FPC2, and FP1-FP3) and reverse (RP) primers used for PCR and RT-PCR amplifications are shown. Restriction sites for HpaI and SacI are indicated. FPC1, FPC2, and RP sequences are common to all known MMTVs. LTR segments of 353, 398, and 426 bp, representing the expression of three novel viruses, RIII/Sa MMTV-1, MMTV-2, and MMTV-3, respectively, were identified in RIII/Sa mice. (B) Evidence for the presence of two distinct MMTVs in the milk and mammary tumors of RIII/Sa mice. Lactating RIII/Sa mice, 8 to 12 days postpartum, were separated from their pups for 6 to 18 h and injected intraperitoneally with 0.2 IU of oxytocin (Sandoz, Basel, Switzerland) 30 min prior to milking. Milk was aspirated with a peristaltic suction pump, diluted 1:20 with a mixture of phosphate-buffered saline (pH 7.4) and 0.15 M EDTA, and centrifuged at 1,000 x g for 10 min. The fat layer and cell pellet were removed, and the milk serum was centrifuged at 20,000 x g for 1 h (31). This partially purified viral pellet, as well as tumor tissue, was used for RNA extraction. To prepare viral cDNA, poly(A)-selected mRNA was incubated with RP (20 pM) at 70°C for 10 min, chilled on ice for 2 min, and then mixed with Promega RT reaction buffer and avian myeloblastosis virus RT (5 U; Promega), and incubated at 37°C for 1 h. PCR amplification was done by mixing 3 to 5 µl of cDNA with deoxynucleoside triphosphates (dNTPs; 5 mM), FPC1 and RP primers (20 pM), and Taq polymerase (1 U; Fisher). The PCR was carried out in a GeneAmp PCR system following a touchdown procedure: 1 cycle of 95°C for 1 min, 60°C for 30 s, and 72°C for 1 min; 1 cycle of 94°C for 30 s, 59°C for 1 min, and 72°C for 1.5 min; 30 cycles of 94°C for 23 s, 58.5°C for 30 s, and 72°C for 1.5 min; and 1 cycle of 94°C for 45 s, 58°C for 45 s, and 72°C for 5 min. MMTV LTR-specific RT-PCR products (indicated by arrows; lanes 2, 4, 6, and 8) were obtained with the primers FPC1 and RP. The PCR results obtained in the absence of RT are shown in lanes 1, 3, 5, and 7. M, milk; T, mammary tumor; L, large cDNA; S, smaller cDNA.

To determine the sequence organization of the cDNAs representing the two viral strains, both milk- and tumor-derived cDNA products were fractionated by preparative (1.5 to 2.0%) agarose gel electrophoresis. The bands of interest were purified with GenEluteMinus ethidium bromide (EtBr) spin columns (Supelco, Bellefonte, Pa.) and precipitated with glycogen. The cDNA products were then cloned into pGEM-T Easy Vector Systems II (Promega) according to the manufacturer's protocol. Positive clones were expanded, and plasmid DNA was isolated with the Wizardplus DNA purification system (Promega). Five individual clones representing each of the four (MS, TS, ML, and TL) RT-PCR products were sequenced with SP6 and T7 promoters and analyzed with the BLAST Program of the National Center for Biotechnology Information. We found that all clones containing the MS or TS cDNAs were identical in their sequence organizations (data not shown). The clones containing the ML product also exhibited identical sequences, and as expected, their 3' sequences differed from the 3' sequences of the MS/TS cDNAs (data not shown). However, the clones containing the TL cDNAs revealed the presence of two different sets of sequences, one of which represented the milk-borne virus RIII/Sa MMTV-2. We concluded that the second set of sequence represented a third viral strain, designated henceforth as RIII/Sa MMTV-3.


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Sequence organization of the ORF of RIII/Sa MMTVs.
 
To determine the sequence of the ORF of the three RIII/Sa viruses, cDNAs were made from milk and tumor tissue RNAs and amplified with the primers FPC2 and RP (Fig. 1A). Gel analysis of the cDNAs showed that both milk and tumor tissue produced approximately 1,300-bp (data not shown) products. Both the milk- and tumor tissue-derived cDNA products were isolated and cloned. Since the sizes of the ORF inserts of different viruses were not expected to differ substantially on agarose gels, we analyzed several clones by double digestion with NotI (two sites in the vector) and HpaI. As expected, digestion with NotI released an insert that, upon digestion with HpaI (this site is conserved in most MMTV 3' ORF sequences and located 22 bp upstream from the primer FPC1), produced two restriction fragments that differed in size by approximately 50 bp (data not shown). This approach led us to identify individual clones containing either the ORF of RIII/Sa MMTV-1 or that of MMTV-2 (Fig. 1). Similar analyses of other clones by SacI and EcoRI (this site is in the vector) digestion resulted in the identification of clones containing either RIII/Sa MMTV-2 or MMTV-3 (Fig. 1). The DNA clones of each viral strain were sequenced (GenBank accession no. AF 136898, AF 136899, and AF 136900, respectively), and the resulting amino acid sequences of their ORFs were determined and compared (Fig. 2). The results show that RIII/Sa MMTV-1 and MMTV-2 were identical in their Sag sequences up to amino acid (aa) 232, and only 2 aa differences were detected between aa 231 and 280. However, within aa 1 to 280, RIII/Sa MMTV-3 differed by 22 or 24 aa. Importantly, the three viruses showed significant variations in amino acids near their C termini (from aa 281 to termination codons). These findings led us to conclude unequivocally that, unlike other native mouse strains, RIII/Sa mice express three specific strains of MMTVs. It should be noted also that none of the RIII/Sa viruses resemble the milk-borne BR6 MMTV of BR6 mice, which has frequently been referred to as RIII MMTV (41).



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  		FIG. 2. Deduced amino acid sequences of the Sags of RIII/Sa MMTV-1, MMTV-2, and MMTV-3. Dots indicate identity. Differences in the number of nucleotides between the Sag protein termination codons and RP (data not shown) resulted in the synthesis of cDNAs of different sizes (L and S), as seen in Fig. 1.

In order determine whether any of the three RIII/Sa ORFs represented endogenous proviruses, primers (FP1, FP2, and FP3) specific to each of the MMTV strains were designed (Fig. 1) from the sequence data we obtained earlier by sequencing the three RT-PCR products synthesized with the FPC1, FPC2, and RP primers (Fig. 1). As shown in Fig. 3A, FP3 and RP primers amplified an approximately 400-bp product from the liver DNA of RIII/Sa (lane 2) and C57BL mice (lane 5), but not from liver DNA of RIIIS/J (lane 3) and BALB/c mice (lane 4). In contrast, no amplification product could be detected from any of the four mouse strains when FP1, FP2, and RP primers were used (Fig. 3A, lanes 7 to 10 and 12 to 15). These observations clearly indicate that while RIII/Sa MMTV-3 is an endogenous virus, both RIII/Sa MMTV-1 and MMTV-2 are exogenous viruses. In view of the fact that our RIII/Sa MMTV-3 provirus is not present in BALB/c mice, which carry Mtv-6, -8, and -9 proviral sequences, but is present in C57BL mice, which carry Mtv-17, in addition to Mtv-8 and -9 proviruses (19), we thought that RIII/Sa MMTV-3 most likely represented Mtv-17. To support this view, we compared the pattern of MMTV LTR-specific PvuII restriction fragments produced from the genomic DNA of RIII/Sa mice with those produced from the genomic DNA of a number of known Mtv-17-carrying mouse strains: DBA2/J, LP/J, AKR/J, C58/J, C57BL/6J, and CBA/J. Because RIIIS/J, like RIII/Sa, is a subline of RIII mice, we also included this mouse strain in the study. Our rational for the choice of PvuII over the traditional EcoRI (19) to analyze RIII/Sa mouse DNA was the realization that in some mouse strains, such as RIII/Sa (30), EcoRI produces cell-virus junction fragments of similar sizes from two or more endogenous proviruses, and thus the identification of the virus on the basis of fragment size becomes problematic (11). As shown in Fig. 3B, our results clearly show that PvuII produced, from the genomic DNA of RIII/Sa mice, a restriction fragment (lane 8), the size of which was similar to those Mtv-17-specific restriction fragments expected to be produced from our control group of mice (Fig. 3B, lanes 1 to 6). Interestingly RIIIS/J mice did not show the presence of this provirus in their genome (Fig. 3B, lane 7). Since the C-terminal sequences of the RIII/Sa MMTV-3 Sag were found to be homologous to the published C-terminal sequences of the Sag of Mtv-17 and Mtv-23 (see Fig. 6), we concluded from the Southern hybridization data that the origin of RIII/Sa MMTV-3 is Mtv-17. It should be pointed out that this virus may not represent the so-called RIIIf MMTV, which is known to be expressed only in older animals (23).



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  		FIG. 3. (A) PCR evidence for the presence of RIII/Sa MMTV-3-specific sequences in the genomic DNA of RIII/Sa (lane 2) and C57BL (lane 5) mice, but not in the genomic DNA of RIIIS/J (lane 3) and BALB/c mice (lane 4). RIIIS/J, C57BL, and BALB/c mice were obtained from the Jackson Laboratory and bred at the Medical College of Georgia. Liver DNA was isolated (29) and subjected to PCR amplification with RIII/Sa MMTV-3, MMTV-2, and MMTV-1 ORF-specific primers (FP3, FP2, FP1, and RP; see Fig. 1A). DNA clones containing the Sag sequences of RIII/Sa MMTV-2 (lane 6) and MMTV-1 (lane 11) were used as positive controls. Note that neither RIII/Sa MMTV-2 (lanes 7 to 10) nor MMTV-1 (lanes 12 to 15) is present in the genome of the four strains of mice that were examined. (B) Patterns of restriction fragments produced by PvuII from the genomic DNA of a number of mouse strains, all of which, except RIII/Sa, were obtained from the Jackson Laboratory. Ten to 15 µg of liver DNA was digested overnight with restriction enzyme PvuII under conditions recommended by the supplier (Promega), size fractionated by electrophoresis through 0.8% agarose, and transferred to a nitrocellulose filter. The filter was then hybridized with an MMTV LTR probe labeled with [{alpha}-32P]dCTP by using an Oligolabeling kit (Pharmacia) and exposed to X-ray film. Note that the DNA digests from RIII/Sa mice, but not from RIIIS/J mice, show an Mtv-17-specific restriction fragment (arrow).



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  		FIG. 6. Alignment of the last 50 C-terminal amino acid sequences of three groups of MMTVs that bear homologies to the three viruses of RIII/Sa mice, RIII/Sa MMTV-1, MMTV-2, and MMTV-3. The amino acid sequences of BR6-MMTV have been used as a reference, since this virus has been characterized as the milk-borne virus of RIII mice. Dots represent identical amino acids, and dashes represent the absence of a residue. Sequence information was obtained from a compilation of the sequences that were published by several groups of investigators. Note that none of the RIII/Sa MMTVs resembles BR6 MMTV.


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Expression pattern of RIII/Sa MMTVs.
 
In view of the fact that MMTV expression in mammary glands and mammary tumors is intimately linked to viral pathogenesis, and because we found that RIII/Sa mice express three different strains of MMTVs, we investigated the pattern of expression of these viral strains in target tissues. Total or poly(A)-selected RNAs obtained from early mammary tumors and mammary glands of both lactating and nonlactating mice of various ages, as well as milk samples collected from the stomachs of young pups, were examined by RT-PCR using primers specific to each of the viral strains. Representative data of our experiments are shown in Fig. 4. Of the 12 mammary tumors tested, RIII/Sa MMTV-2 was expressed in all tumors (for example, see lanes 2, 5, and 8 in Fig. 4A), whereas RIII/Sa MMTV-1 expression was not detected in three tumors (examples of negative and positive results are shown in lane 3 and in lanes 6 and 9, respectively). However, both virus strains were found to be present in various amounts in the milk and mammary glands of 12 young and 10 older mice that we tested. During the course of this work, we were able to examine only two mammary tumors of BALB/c mice that were foster-nursed on a third-parity RIII/Sa mother. The tumors were found to express both RIII/Sa MMTV-1 and MMTV-2 (Fig. 4A, lanes 11 and 12). This observation suggests that both MMTVs are infectious to BALB/c mice and that at least one of the viruses must have played a role in tumor development. Interestingly, we could not detect by RT-PCR the presence of RIII/Sa MMTV-1- or MMTV-2 ORF-specific sequences in mammary glands and milk of RIII/SJ mice (data not shown). As a matter of fact, no MMTV RNA was detected in the milk of RIII/SJ mice by Northern blotting (Fig. 4F). This finding is consistent with the observation made by the Jackson laboratory that RIIIS/J mice do not develop early mammary tumors (www.informatics.jax.org/menus/strain-menu.html). It is difficult to explain why RIIIS/J mice are currently free of any exogenous MMTV, since they were known to express milk-borne MMTV as early as 1962 and as late as 1995 (1, 9, 33, 37). The Jackson Laboratory derived the RIIIS/J mouse strain by cross-breeding between an exogenous MMTV-expressing parental RIII female mouse and a male SEC/1ReJ mouse (34), and thus MMTV production should not have been affected in RIIIS/J mice. We have observed that low-parity RIII/Sa mothers express much smaller amounts of MMTV particles than high-parity mothers, and thus we use pups from high parity mothers (3rd parity or more) for breeding so that the mice of succeeding generations maintain high virus production. The tendency of RIII mice to lose MMTV production had been noted as early as 1962 (1). We believe that RIIIS/J mice lost milk-borne MMTV because the Jackson Laboratory may not have always used pups from high-parity mothers for routine breeding.



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  		FIG. 4. (A) Patterns of the expression of the RIII/Sa MMTV-1 (lanes 3, 6, and 12) and MMTV-2 (lanes 2, 5, 8, and 11) ORF in RIII/Sa and BALB/cfRIII/Sa mouse mammary tumors. RIII/Sa MMTV-1 (1) and MMTV-2 (2)-specific primers were used, in the presence (+) and absence (-) of RT for the synthesis of cDNAs. (B to E) Evidence for the expression of RIII/Sa MMTV-3 (Mtv-17) RNA in mammary glands (B, lanes 1 to 3), mammary tumors (B, lanes 4 and 5), spleens (B, lanes 8 and 9), and milk (E, lanes 1 to 5 and 7) from RIII/Sa mice. RT-PCR was also done with RNA obtained from one lactating mammary gland (B, lane 6) and one milk sample (E, lane 6) from a C57BL/10J mouse and one lactating mammary gland (B, lane 7) and one spleen tissue sample (B, lane 10) from an RIIIS/J mouse. Panel C represents the results of PCRs done with the samples used in panel B in the absence of RT; the use of primers common to all MMTV ORFs produced cDNA from all samples (D). (F) Northern blot hybridization for the detection of viral RNA in stomach milk of RIIIS/J and RIII/Sa mice. Virus was isolated by using the milk pellet obtained from the stomachs of 2- to 4-day-old pups (14). RNA was prepared and fractionated (10 µg/lane; migration from left to right) on a 1% formaldehyde gel. The gel was stained with EtBr prior to blotting in order to ensure RNA integrity and equivalent sample loading (panel Fa). Hybridization was done with a full-length MMTV probe (30). Arrowheads (panel Fa) indicate the 28S and 18S rRNA; the arrow in panel Fb points to full-length MMTV-RNA. Short- and long-exposure films of the same blot are shown in panels Fb and Fc, respectively.

With regard to the expression of RIII/Sa MMTV-3 (Mtv-17) in normal tissues of RIII/Sa mice, RT-PCRs carried out with Mtv-17 ORF-specific primers, showed the synthesis of specific cDNAs from RNA prepared from nonlactating or lactating mammary glands (Fig. 4B, lanes 1 to 3), mammary tumors that were used as controls (Fig. 4B, lanes 4 and 5), and from some spleens (Fig. 4B, cDNA+, lane 8; cDNA-, lane 9). Lactating mammary glands of C57BL/10J mice were also positive for the ORF transcript (Fig. 4B, lane 6). As anticipated, RIIIS/J mice were negative for the expression of RIII/Sa MMTV-3 ORF-specific transcript in their mammary glands (Fig. 4B, lane 7). None of the RNA samples shown in Fig. 4B when similarly tested in the absence of RT, produced any cDNA(Fig. 4C), indicating that our positive RT-PCR results were the products of RNA instead of possible contaminating genomic DNA. The 10 RNA samples used in Fig. 4B were also subjected to RT-PCR amplification with a primer set that represented a segment of the ORF common to all endogenous and exogenous MMTVs. The results show that all samples, including RIIIS/J, produced cDNA (Fig. 4D), suggesting that those tissues that were negative for Mtv-17 ORF do express other endogenous MMTV ORF. RT-PCR analyses showed that the milk pellets obtained from the stomachs of RIII/Sa mouse pups (Fig. 4E, lanes 1 to 5 and 7), but not the milk pellets from the pups of C57BL mice, that express Mtv-17 ORF (for example, see Fig. 4E, lane 6), contained ORF RNA specific to RIII/Sa MMTV-3. Taken together, our results suggest that, in general, RIII/Sa mice express two strains of exogenous infectious MMTVs and the transcript of Mtv-17. We do not know how Mtv-17 expression is linked to mammary tumorigenesis in RIII/Sa mice. It is possible that (i) Mtv-17 may be expressed as a functional provirus and produce infectious virus particles like Mtv-1, Mtv-2, and Mtv-48; or (ii) RIII/Sa MMTV-3 may be a recombinant virus involving Mtv-17 Sag and RIII/Sa MMTV-1 and/or MMTV-2. Further work is needed to know the biological significance of Mtv-17 Sag expression in RIII/Sa mice.


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int mutation.
 
The hallmark of MMTV-induced mammary tumorigenesis is the activation of a number of oncogenes, primarily int-1/Wnt-1 and int-2/Fgf 3 oncogenes, as a consequence of proviral integration in the vicinity of the target genes. Since we found that three different strains of MMTVs are expressed in mammary tumors of RIII/Sa mice, we sought to determine a priori which of the viruses could be involved in mammary tumor development, because of the fact that the presence of different types of virions in a tumor does not necessarily imply that each virus type caused an insertion mutation of ints. Further, there is some evidence that an infectious virus, such as MMTV-SW, may not always induce mammary tumors in its natural host (16). Our strategy to characterize the oncogenic MMTVs was to clone MMTV int-1/Wnt-1 or MMTV int-2/Fgf 3 junction fragments and determine the sequences of the viral LTR ORF. Genomic DNA from 12 mammary tumors of RIII/Sa mice were prepared, digested with BglII or EcoRI, and analyzed by Southern hybridization with int-1/Wnt-1- or int-2/Fgf3-specific probes. Results from 7 of the 12 tumors tested are shown in Fig. 5C to E. Two of the 12 tumors (tumors 2 and 6) showed rearranged restriction fragments with hybridization intensities similar to those of the unrearranged fragments (Fig. 5C, lane 6, and E, lane 2). The sizes of the MMTV int-1/Wnt-1 and MMTV int-2/Fgf3 junction fragments were 8.1 and 5.2 kb, respectively. Two other tumors also produced rearranged restriction fragments that hybridized poorly with the probes (arrows in Fig. 5C, lane 1, and E, lane 3). In order to determine the orientation of proviral integration in these tumors, int-specific probes were stripped off the filter and rehybridized with an MMTV env or LTR probe (data not shown). These experiments allowed us to know the orientations of proviral integrations associated with the 8.1-kb (BglII/int-1C) and 5.2-kb (EcoRI/int-2S) junction fragments (Fig. 5B). It should be noted that the orientation of proviral integration in one of the tumors within the int-1/Wnt-1 locus is unusual (Fig. 5B), since the transcriptional orientation of MMTV provirus in most mammary tumors, except in some rare tumors, has been found to be away from the transcritional orientations of both int-1/Wnt-1 and int-2/Fgf3 (4).



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  		FIG. 5. Southern blot hybridization for the detection of int-1/Wnt-1 and int-2/Fgf3 rearrangements in the genomic DNA of mammary tumors from RIII/Sa mice. (A) Structure of an RIII/Sa MMTV provirus showing the location of the viral LTRs and the BglII (Bg) and EcoRI (E) restriction sites. (B) Linear maps of int-1/Wnt-1 and int-2/Fgf3 and the locations of the 1C and 2S probes (heavy lines beneath the int maps). The solid boxes represent coding exons for the respective int genes. S, SacI; Bm, BamHI. (C to D) High-molecular-weight DNA was isolated from tumors (30), and 10 to 15 µg of DNA was digested overnight with restriction enzyme BglII or EcoRI and analyzed by Southern hybridization with either the int-1/Wnt-1C or int-2/Fgf3-2S probe (4, 27). Four of the seven tumors had MMTV-induced rearranged ints; two of the restriction fragments (indicated by stars in (C, lane 6; E, lane 2) hybridized with the probes very strongly, while the intensity of hybridization of the other two rearranged fragments was very weak (arrows in panel C, lane 1, and panel E, lane 3). The sizes of the unrearranged BgIII/int-1C (panel C), EcoRI/int-1C (panel D), and EcoRI/int-2/Fgf3-2S (panel E) fragments, as expected, were 10.2, 8.0, and 10.0 kb, respectively. Further analysis of the rearranged fragments of 8.1 and 5.2 kb with an MMTV LTR probe confirmed that they contained LTR sequences (data not shown). The locations of the MMTV proviruses that caused disruption of the int domains (C, lane 6, and E, lane 2) by integration and the transcriptional orientations of the virus are shown above the linear maps of the int genes by arrowheads (B).

To evaluate which of the three RIII/Sa MMTVs caused insertional mutation in tumors 2 and 6, the 8.1- and 5.2-kb bands were isolated from preparative agarose gels by electroelution, concentrated by extraction with 2-butanol/phenol-chloroform and ethanol precipitation, and further purified by passage through an Elutip-d column (29). DNA was ligated to pGEM-3z Vector (Promega) at the BamHI or EcoRI cloning sites according to the manufacturer's protocol. Clones containing inserts were selected by blue/white color screening, and amplified DNA was extracted and analyzed by Southern blotting with MMTV LTR and Wnt-1C or int-2/Fgf3-2S probes. It was found that the cloned 8.1- and 5.2-kb fragments contained LTR-specific sequences as expected (data not shown). The ORF-specific sequences associated with the clones were PCR amplified with the primers FPC1 and RP (Fig. 1), cloned into pGEM-T Easy Vector Systems II, and sequenced. RIII/Sa MMTV-2 was identified to be the virus that was associated with both junction fragments and thus was most likely to have induced both tumors. One recent study has determined the Sag sequence of a provirus (GenBank accession no. AF 071010) that was cloned from the mammary tumor of an RIII/Sa mouse (28). The Sag of this virus bears very close homology to the Sag of RIII/Sa MMTV-2. Taken together, it appears that RIII/Sa MMTV-2 may be a major determinant for mammary tumorigenesis in RIII/Sa mice. A large panel of RIII/Sa mammary tumors must be analyzed to confirm this and to determine whether RIII/Sa MMTV-1 and/or MMTV-3 participates in the activation of some ints. Such a study however, presents considerable challenges, but may provide valuable information on the role of multiple MMTVs in mammary tumor development not only in RIII/Sa mice but also in other mouse models.


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Identity of RIII/Sa MMTV Sags with the Sags of other MMTVs.
 
Sequence analyses of the Sags of some 35 other endogenous and exogenous MMTVs have resulted in the groupings of most MMTV strains into seven major families on the basis of the composition of their carboxyl-terminal amino acids (41). Alignment of the sequences of the 44 C-terminal amino acids of the RIII/Sa MMTV-1 Sag with the known sequences of the C termini of other MMTV Sags revealed that this virus belongs to a group of MMTVs with T-CR Vß-2 specificity: BALB2, C4, Mtv-DDO, II-TES 2, CS/Mtv-48, BALB/cV, and C3H-K (Fig. 6). Despite this Vß-2 specificity, these viruses show diverse biological characteristics. For example, the origin of C3H-K virus is the kidney tumor of a BALB/cfC3H mouse, yet unlike C3H MMTV, it does not cause mammary tumors (32, 39). The Sag sequences of this virus differ significantly from that of C3H MMTV, but are almost identical to the Sag sequences of oncogenic BALB2 and BALB/cV MMTVs. The host animals of the BALB2, C4, BALB/cV, and C3H-K viruses are BALB/c mice (7, 18, 32, 38, 39). However, most colonies of BALB/c mice are known to be free of exogenous virus (21, 23, 24), but are highly susceptible to infection by various exogenous MMTV strains (23), including RIII/Sa MMTV-1 (Fig. 4A; lanes 11 and 12). Three endogenous proviruses, Mtv-DDO, CS/Mtv-48, and CS/Mtv-51, also share the same Vß-2 specificity with the other exogenous viruses of this group, but only CS/Mtv-48 has been shown to be infectious to BALB/c mice (17, 26).

The RIII/Sa MMTV-2 virus shares very close homology in the C terminus of the Sags of three other biologically different MMTV strains, FM, SHN, and Mtv-RCS (20, 35, 42) (Fig. 6). Both SHN and FM are tumorigenic, but the FM virus is exogenous, while the SHN virus is an infectious analogue of an endogenous provirus, Mtv-4. Similar to SHN, Mtv-RCS is an endogenous virus, but unlike SHN, Mtv-RCS does not produce infectious virus particles, although its ORF is highly expressed in spontaneously developed reticular cell sarcomas (RCS) of SJL mice (35). It is quite striking that the TCR Vß specificity of Mtv-RCS virus differs not only from the SHN and FM viruses, but the other 33 different MMTV strains (41); Mtv-RCS Sag is reactive only to Vß-16 T cells. While the Sag of SHN-MMTV interacts with Vß-7-, Vß-8.1-, Vß-8.2-, and Vß-8.3-specific T cells, FM virus infection causes deletion of Vß-2, Vß-6, Vß-8.1, Vß-8.2, Vß-8.3, and Vß-14. Thus the FM virus shares Vß-14 specificity with some highly oncogenic GR, C3H, and BR6 MMTVs, as well as with other less-oncogenic viruses, despite the fact that the extreme C-terminal Sag sequences of FM virus are quite different from the sequences of the respective Vß-2- and Vß-14-specific viruses. It appears, therefore, that similarity in amino acid sequences of the ORF or TCR Vß specificity among different MMTVs may not be a good predictor of their pathobiology. We have recently found that infection from milk-borne RIII/Sa MMTVs leads to the deletion of both Vß-2- and Vß-8-specific T cells (35a). Based on the homology of Sags, we predict that RIII/Sa MMTV-1 and MMTV-2 would be specific for TCR Vß-2 and Vß-8, respectively. Further studies involving the cloning of individual RIII/Sa MMTV strains and testing their TCR Vß specificity are needed to confirm our prediction. The availability of such cloned viruses should also allow us to determine directly the relative effectiveness of these viruses in mammary tumor development.


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Nucleotide sequence accession numbers.
 
The sequences portrayed in Fig. 2 have been reported to GenBank under accession no. AF136898, AF136899, and AF136900.


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ACKNOWLEDGMENTS
 
This work was partially supported by grants from the American Cancer Society (N.H.S.) and the National Institutes of Health (T.A.G).

We thank R.-B. Markowitz for editorial assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912. Phone: (706) 721-7657. Fax: (706) 721-7915. E-mail: nsarkar{at}mail.mcg.edu. Back

{dagger} Present address: Center for Liver Diseases, Owaisi Hospital and Research Center, Kanchanbagh, Hyderabad-500058, India. Back


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Journal of Virology, January 2004, p. 1055-1062, Vol. 78, No. 2
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.2.1055-1062.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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