Type-specific determinants on proteins of an endogenous C3H mouse mammary tumor virus (MMTV) distinguish this virus from highly oncogenic exogenous MMTVs

The genetically transmitted endogenous MMTV isolated from C3H mice after removal of the milk-transmitted virus by foster nursing is designated C3Hf MMTV to distinguish it from the highly oncogenic milk-transmitted exogenous virus designated C3H MMTV. We have isolated a MMTV-expressing C3Hf mammary tumor cell line which has no exogenous proviral sequences detectable by analysis of DNA fragments generated by Pst I restriction endonuclease. This cell line produced sufficient C3Hf MMTV to allow purification of the major proteins and an antigenic comparison of this virus with highly oncogenic exogenous MMTVs from C3H, GR, and RIII strains of mice. The envelope glycoproteins, gp52 and gp36, purified from the C3Hf MMTV, were found to have both group- and type-specific reactivities. Only C3H MMTV gave incomplete competition in the gp36 assay and, therefore, could be distinguished from C3Hf, RIII, and GR MMTVs which gave complete competition. Unique antigenic determinants exist on the gp52 of C3Hf MMTV since it is the only virus to give complete competition in the gp52 radioimmunoassay. GR MMTV competed only 60%, whereas C3H and RIII MMTVs gave 80% competition. This was the first demonstration that RIII and C3Hf MMTVs were immunologically distinct. Only group-specific reactivity was found with the gag-coded MMTV p27; however, group and class antigenic determinants were found on the gag-coded MMTV p10.     

Coexistence of the mouse mammary tumor virus (MMTV) major glycoprotein and natural antibodies to MMTV in sera of mammary tumor-bearing mice.

Sera of mammary tumor-bearing mice contain the major envelope glycoprotein (gp52) of mouse mammary tumor virus (MMTV) and autogenous antibodies to MMTV. Although antibodies to MMTV are readily demonstrable in sera of both tumor-free and tumorbearing animals, detection of the viral glycoprotein appears to be dependent on the presence of a palpable mammary tumor. MMTV gp52 was detected both as the free protein and in a high molecular weight complex as demonstrated by velocity sedimentation centrifugation. MMTV gp52 was also detected in both mammary tissue and lymph nodes of female C3H/HeN mice. The reproductive organs of the male C3H/HeN mice, such as the vas deferens and vesicular, coagulating, and prostate glands, contained gp52, while no MMTV gp52 could be demonstrated in the testes, epididymis, or preputial glands. This antigen was not found in any tissues of male or female BALB/c mice.     

Generation of a mouse mammary tumor virus (MMTV) pseudotype of Kirsten sarcoma virus and restriction of MMTV gag expression in heterologous infected cells.

C3H mouse mammary tumor cells producing mouse mammary tumor virus (MMTV) were cocultivated with nonproducer mouse cells (KNIH) transformed by Kirsten sarcoma virus (KiSV). These cocultivated cells were then treated with mitomycin C and overlayed onto human embryonic skin and muscle cells. The virus resulting from this cocultivation could be titrated in a focus-forming assay on Fischer rat embryo (FRE) cells exhibiting one-hit kinetics. Furthermore, focus formation on FRE cells was neutralized specifically by antiserum directed against MMTV and the major MMTV external glycoprotein gp52, but not against a broadly reactive antiserum directed murine leukemia virus (MuLV) gp70 and MMTV gp36, p27, p14, and p10. These results demonstrate the generation of a KiSV(MMTV) pseudotype and further demonstrate that gp52 is a target antigen for neutralization of MMTV. This pseudotype possessed a wide host range, transforming cells of human, rat, mouse, mink, and rabbit origin. MMTV but not MuLV antigen expression was demonstrated in the KiSV(MMTV) pseudotype-infected cells. Analysis of intracellular MMTV protein synthesis in these in vitro-infected cells has indicated that the low yield of extracellular MMTV produced by the transformed cells may be the result of the poor expression of the MMTV gag precursor polyprotein relative to the expression of the env gene polyprotein. These studies thus provide the basis for an in vitro infectivity assay for neutralization and host range studies of MMTV.     

Mouse mammary tumor virus (MMTV) infection in SWISS and RIII mice

Summary Host-virus relationships were examined in mice from the two mouse mammary tumor virus (MMTV)-infected strains SWISS MB+ and RIII, which harbour the same MMTV variant, and from the derived sublines Swiss MB-and RIIIf, which were freed of milk-borne MMTV by foster-nursing. These two strains are not phylogenetically related, the SWISS strain bearing the endogenousMtv-3 locus in its DNA. In RIII and SWISS MB+ mice, the incidence of early mammary tumors, which was of 96% and 8%, respectively, was correlated to the level of MMTV expression in milk. In the SWISS MB-line, a non-coordinate expression of the provirus associated with theMtv-3 locus was observed in the mammary glands, the salivary glands and the spleen. This expression was not tumorigenic and was characterized by the presence of the p28gag antigen and the absence of the gp52env antigen, except, however, in mammary glands of elder mice where traces of gp52 were found. In the mammary glands of SWISS MB+ mice, the expression of theMtv-3 locus was masked by large amounts of antigens resulting from exogenous virus expression. RIIIf mice were MMTV-negative.Viral antigens coexisted with anti-MMTV antibodies in the serum of infected and tumor-bearing mice, but not in the form of immune complexes as verified by a method that allowed to detect specific antigen-containing-soluble immune complexes. An anti-MMTV serum reactivity was also detected in SWISS MB-and RIIIf mice. However, the serum response was higher in the two SWISS lines than in the two RIII lines. Except in tumor-bearing mice, the anti-MMTV response was not significantly modified by the presence of exogenous virus and thus resulted essentially from exposure to endogenous MMTV expression. In experimental infection studies, RIII mice were more susceptible to MMTV infection than SWISS mice. The correlation between resistance to MMTV infection and serum response to endogenous MMTV expression, suggests that the non-tumorigenic expression of an endogenous provirus can protect at least partially, against exogenous MMTV infection.     

Role of a heterologous retroviral transport element in the development of genetic complementation assay for mouse mammary tumor virus (MMTV) replication

The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple “tissue specific” and “hormone inducible” promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.     

Possible pathways of circulation of human endogenous retrovirus similar to mouse mammary tumor virus

It is shown that polypeptides which are immunologically related to gp52 mammary tumor virus are found in T and B peripheral blood lymphocytes in all breast cancer patients, in children with B-cell lymphosarcomas, and in B lymphocytes of some healthy donors. These proteins are not found in patients with tumors of other sites. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp.554–556, November, 1995 Presented by Yu. N. Solov'ev, Member of the Russian Academy of Medical Sciences     

Presence of antibodies reacting specifically with structural proteins of mouse mammary tumor virus (MMTV) among antibodies composing circulating immune complexes in breast cancer patients

All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 4, pp. 475–477, April, 1988.     

Antisense downregulation of a mouse mammary tumor virus activated protooncogene in mouse mammary tumor cells reverses the malignant phenotype

Activation of the protooncogene Wnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice. Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state. Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuous Wnt-1 expression for their ability to form tumors in vivo. To address this question, we constructed antisense and sense Wnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenous Wnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and sense Wnt-1 RNAs, changes in endogenous Wnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cells in vitro and in vivo were also examined. Our results show that (1) the levels of the expression of endogenous Wnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisense Wnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors ( approximately 0.2 cm in 16 weeks) compared to the tumors ( approximately 2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest that Wnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.     

Murine mammary tumor virus (MuMTV) infection of an epithelial cell line established from C57BL/6 mouse mammary glands.

An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by routine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTV-related DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 × 10⁵ virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect any morphological or growth pattern changes in C57MG cells following infection with MuMTV.     

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.     

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines.

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10? to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.     

Human endogenous retrovirus IDDMK(1,2)22 and mouse mammary tumor virus superantigens differ in their ability to stimulate murine T cell hybridomas

Recently, a newly identified human HERV-K18 like endogenous retrovirus (IDDMK(1,2)22) has been associated to the etiology of type I diabetes (IDDM). Although the exact mechanism remains unclear, it was postulated that the 3' end ORF product of the env gene of IDDMK(1,2)22 would trigger a V beta 7-specific human T cell expansion leading to their infiltration in the pancreas of afflicted patients and to the autoimmune destruction of the insulin-producing beta cells. Since then, such superantigen (SAg)-like activity as well as the association between the IDDMK(1,2)22 virus and IDDM pathogenesis have been challenged. To further characterize functionally the putative IDDMK(1,2)22-encoded SAg, we have cloned from human DNA the identical 462bp ORF sequence originally described. The IDDMK(1,2)22 ORF fragment was transfected in the same human B cell line (Raji) originally used as APC to demonstrate the V beta 7 specificity. The immunostimulatory potential of IDDM ORF was tested on murine T cell hybridomas and compared to the well-characterized mouse mammary tumor virus Mtv7 SAg transfected in the same conditions. A panel of 16 T cell hybridomas encompassing 14 different V betas was analyzed. We have failed to detect IDDMK(1,2)22-induced IL-2 production from any of these hybridomas, even those bearing the murine V beta 1 mV beta 1, V beta 4 or V beta 10 TcR beta chains which are most closely related to the human V beta 7 (hV beta 7). Our results suggest that IDDMK(1,2)22 ORF is devoid of superantigenic activity as defined by classical criteria.