Further evidence for the protein coding potential of the mouse mammary tumor virus long terminal repeat: nucleotide sequence of an endogenous proviral long terminal repeat

The 3′ half of an endogenous mouse mammary tumor virus from a C3H mouse was cloned in the Charon 4A vector phage. A comparison of the proviral clone with previously published endogenous mouse mammary tumor virus restriction maps identified it as endogenous unit II (J. Cohen and H. Varmus, Nature [London] 278:418-423, 1979), which is present in all inbred mouse strains derived from the original Bagg albino × DBA cross. The nucleotide sequence of the unit II long terminal redundancy (LTR) was determined and compared with the sequence previously determined for the exogenous C3H virus LTR (Donehower et al., J. Virol. 37:226-238, 1981). Virtually all sequence differences between the two LTRs were base substitutions. The total amount of sequence divergence was 6.6%. The large open reading frame reported previously in the exogenous LTR was preserved in the endogenous LTR. In addition, the pattern of sequence divergence was highly nonrandom with respect to the putative amino acid codons of the two open reading frames. Most of the base substitutions in this region resulted in silent or conservative amino acid codon changes. The nonrandom divergence pattern indicates that selective forces are operating on this segment of DNA and argues that the putative protein is functional in the life cycle of mouse mammary tumor virus. Possible roles for the protein and its mode of expression are discussed.     

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.     

Covalent and noncovalent receptor-glucocorticoid complexes preferentially bind to the same regions of the long terminal repeat of murine mammary tumor virus proviral DNA

Dexamethasone 21-mesylate, an irreversible antiglucocorticoid in HTC cells, forms a covalent receptor-steroid complex which can be activated in cell-free systems. The molecular basis of its antiglucocorticoid activity is unknown; it might result from altered DNA sequence preferences and/or affinities of the covalent receptor-steroid complex. To test this hypothesis, the affinities of both covalent receptor-antagonist and noncovalent receptor-agonist complexes for defined DNA sequences were measured in a DNA binding competition assay. This assay requires neither purified complexes nor large quantities of DNA, yet it provides quantitative comparisons of the affinities of different double-stranded DNAs for binding receptor-steroid complexes. In this assay, activated covalent receptor-dexamethasone 21-mesylate complexes in crude cytosol bound to calf thymus DNA and cloned subregions of the long terminal repeat (LTR) of murine mammary tumor virus (MMTV) proviral DNA with approximately the same relative affinities as did noncovalent receptor-dexamethasone complexes. Both types of complex exhibited similar orders of preferential binding to DNA sequences. LTR subregions, as well as the entire LTR, were 2-20 times more potent competitors than calf thymus DNA. Cloned sequences from the 3' terminus of the LTR were more effective competitors than either the entire LTR or comparably sized DNAs from the 5' terminus. The DNA sequences with the greatest affinities for both covalent and noncovalent complexes are located within the region of -221 to -67. These studies support the theory that recognition by regulatory elements of specific DNA sequences upstream of responsive genes is an integral step of hormone action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.     

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.     

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3'' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.     

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.