Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.     

Genetic relationship between the Mus cervicolor M432 retrovirus and the Mus Musculus intracisternal type A particle

The genetic relationship between the retrovirus-like intracisternal type A particle (IAP) from Mus musculus and the novel retrovirus (M432) from M. cervicolor has been determined by heteroduplex and restriction endonuclease analyses of molecular clones of the respective genomes. We have found a major homology region (3.7 kilobase pairs) which probably begins near the 3' end of the M432 gag gene, spans the pol gene, and ends in the env gene. A second region (0.6 kilobase pairs) of weak homology was also observed adjacent to the 3' long terminal repeats of the respective genomes. The IAP genome is well conserved in the cellular DNA of all species of the genus Mus. In contrast, cellular DNA sequences related to the 5' end of the M432 genome, which shares no homology with the IAP genome, are found only in M. cervicolor and the closely related species M. cookii. These results suggest that the infectious M432 retroviral genome arose as a result of a recombinational event(s) between the IAP genome and another, as yet unidentified, class of retrovirus-related sequences or other cellular sequences.     

An antigen related to the mouse mammary cancer virus env gene product, detected in human lymphocytes, is associated with human breast cancer]

Expression of DNA sequences homologous to sequences of env gene of mouse mammary tumor virus (MMTV) in the lymphocytes of patients with breast cancer and in subjects at a high risk of breast cancer has been reported. Antigen analogous to envelope protein gp52, product of MMTV env gene, is detected in T lymphocytes of virtually all patients with breast cancer and extremely rarely in T cells of controls, where its expression is confined to B cells. For explaining such unexpected results, we studied the molecular basis of this antigen synthesis. Specific PCR products were obtained using primers to gp52-coding region of MMTV env gene. One of them (957 nucleotides) was used as a probe for hybridization of DNA and RNA from lymphocytes of patients with breast cancer and controls. This sequence was hybridized with 90% frequency with genome DNA of breast cancer patients and with 85% frequency with genome RNA of such patients, which is almost 4-fold more than in the controls (patients with gynecological tumors or donors). These results correlate with the frequency of detection of the studied antigen in patients with breast cancer and control group patients.     

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.     

Molecular cloning of retrovirus-like genes present in multiple copies in the Syrian hamster genome.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3' end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.     

Nucleotide sequence of the intracisternal A-particle genome inserted 5'' to the interleukin-3 gene of the leukemia cell line WEHI-3B.

The nucleotide sequence of the intracisternal A-particle genome IAP-IL3 is presented. This IAP element was found to have inserted upstream of the promoter of the interleukin-3 gene of the leukemia cell line WEHI-3B. IAP-IL3 is 5095 bp in length, with identical long terminal repeats (LTRs) of 337 bp. The LTRs show many of the conserved sequence elements identified in other retroviruses. Comparison with other available sequences of IAP genomes indicates that IAP-IL3 is a deleted type I element. It carries a deletion covering the 3' end of the putative IAP gag gene and extending into the 5' end of the putative IAP pol gene. IAP-IL3 has extensive sequence homology with an IgE-binding factor cDNA and evidence is presented indicating that it was derived from a member of the mouse IAP sequence family. Comparison between the pol region of IAP-IL3 and other retroviruses suggests that IAP-IL3 is most closely related to type B and type D retroviruses.     

Effect of 5-azacytidine on expression of DNA sequences homologous to ENV gene of murine mammary tumor virus in normal human lymphocytes

Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.     

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.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.     

Nonrandom distribution of MMTV proviral sequences in the mouse genome.

Integrated sequences of mouse mammary tumor virus (MMTV) have been localized in the genomes of five inbred mouse strains (Balb/c, C3H, DBA/2, A.TH, 129-SV) and one mammary tumor cell line (GR). Two major classes of MMTV sequences have been detected in mouse DNA fractions as obtained by Cs2SO4/BAMD (3,6-bis-(acetatomercurimethyl)dioxane) density gradient centrifugation. The first one corresponds to previously described endogenous sequences (Mtv loci), whereas the second one corresponds to endogenous sequences not previously known, and/or recently acquired; in the case of GR cells exogenous sequences may also be present in this class. The genome distribution is somewhat different for the two classes of sequences, the first one being practically only present in the lightest DNA segments of the mouse genome (GC congruent to 38%); the second one being also represented in heavier segments (GC congruent to 43%). This integration pattern suggests that "ancient" endogenous sequences are practically only localized in genome segments of roughly matching composition, whereas exogenous and recently acquired endogenous MMTV sequences may also be present in heavier fractions.     

Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA.     

The genome and the intracellular RNAs of avian myeloblastosis virus

Avian myeloblastosis virus (AMV) is an acute leukemia virus which causes a myeloblastic leukemia in birds and transforms myeloid hematopoietic cells in vitro. We have analyzed RNA from AMV virions and from AMV-transformed producer and nonproducer cells by gel electrophoresis followed by transfer to chemically activated paper and hybridization to several complementary DNA (cDNA) probes. Using a cDNA probe specific for AMV, we identified two RNA species of 7.2 and 2.3 kb, which were present in all AMV-transformed cells and in all AMV virion preparations examined. The 7.2 kb species, which is presumably the genome of AMV, appears to contain the entire retroviral gag gene and at least part of the pol gene, but lacks much (or all) of the env gene. Thus AMV differs from other acute leukemia viruses described to date, since the latter have genomes of 5.5 to 5.6 kb, have only part of the gag gene and lack pol sequences. The smaller RNA does not contain gag-, pol- or env-specific nucleotide sequences but does carry nucleotide sequences from both the 5' and 3' termini of the genome, suggesting that it may be a subgenomic mRNA. Both the 7.2 and 2.3 kb species were associated with the 70S RNA complex in virions. These results suggest that AMV, unlike other acute leukemia viruses, does not express its transforming gene via a gag-related "fusion" protein but rather as a (so far unidentified) protein translated from a subgenomic mRNA.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.