Evidence of recombinant ecotropic provirus integration in thymic lymphomas induced by direct or indirect radiation effects.

Several investigators described the occurrence of ecotropic recombinant proviruses in the DNA of in-vivo or in-vitro propagated radio-induced lymphomas, but such proviruses were never detected in primary tumors. To assess their biological significance in the tumorigenic process, we reinvestigated the presence of new proviruses chiefly in primary radio-induced tumors and in models of radioleukemogenesis which could give additional support for their role. Such models included thymic lymphomas originating after (i) graft of non-irradiated thymuses in thymectomized irradiated mice and (ii) the injection of a B-ecotropic retrovirus (T1223/B) in association with a subleukemogenic dose of irradiation. We report for the first time that new ecotropic proviral sequences are encountered in a significant number (30%) of primary lymphomas induced directly by irradiation or indirectly in non-irradiated thymuses grafted in irradiated hosts. The existence of a 3.5-kbp Kpn1 restriction fragment with ecotropic sequences in the digested DNA of these tumor cells indicates that these new sequences belong to an ecotropic provirus recombinant in the gag-pol region. We observed that most of the primary radio-induced tumors in which novel recombinant provirus could be detected, displayed the integration at a single or at a few sites, demonstrating their clonality with respect to viral integration. The same was observed in thymic lymphomas arising after T1223/B virus injection and irradiation and in in-vivo or in-vitro propagated tumors. Altogether, these data bring the first evidence of the integration of ecotropic recombinant proviral genomes in a significant number of primary radiation induced thymic lymphomas and of their possible role in view of their frequent occurrence in grafted thymomas.

Chromatin structure of hormone-responsive Moloney murine leukemia virus proviruses that contain sequences from mouse mammary tumor virus.

The chromatin structure of chimeric Moloney murine leukemia viruses (M-MuLVs) containing a glucocorticoid response element (GRE) from mouse mammary tumor virus (MMTV) inserted into the long terminal repeat (LTR) was investigated. Nuclear run-on assays indicated that transcription from the chimeric proviruses was induced 2- to 4-fold by dexamethasone. The wild-type M-MuLV 5' LTR contained a DNase I hypersensitive (HS) site at the TATA sequences, as well as four sites in the enhancer region. The chimeric LTRs contained these sites, as well as three additional sites in the MMTV sequences. Two of the MMTV sites were present in the absence of hormone, while one was hormone-induced. In addition, internal MMTV sequences appeared protected from DNase I digestion in the absence of hormone, suggesting bound protein. Hormone treatment resulted in loss of the DNase I protection.

DNA blotting analysis of human retroviruses in cerebrospinal fluid of spastic paraparesis patients: the viruses are identical to human T-cell leukemia virus type-1 (HTLV-1).

The structure of the integrated provirus in cell lines established from the cerebrospinal fluid (CSF) of patients with human T-cell leukemia virus type-1 (HTLV-1)-associated myelopathy (HAM) was analyzed. The digestion patterns with 3 restriction endonucleases, Sac I, Eco RI and PstI, of the proviruses integrated in T-lymphoid cell lines derived from the CSF of 4 HAM patients were similar to those of HTLV-1 from adult T-cell leukemia (ATL) patients. Integrated proviruses in one of these cell lines derived from the CSF were further analyzed in detail with 2 more restriction enzymes, BamHI and Sma I. The results indicate that the retrovirus found in lymphocytes of the CSF from patients with HAM are very similar, if not identical, to HTLV-1 found in the leukemic lymphocytes of ATL patients.

A transcriptionally defective long terminal repeat within an endogenous copy of mouse mammary tumor virus proviral DNA.

Mouse mammary tumor virus proviral DNA is endogenous to most inbred strains of mice but in many strains is not transcriptionally active. This inactivity may be due to defects in the proviruses themselves or to position effects mediated by DNA sequences flanking the proviral units. The transcriptional competence of long terminal repeats (LTRs) derived from endogenous proviral DNA at genetic loci Mtv-8, Mtv-9, and Mtv-17 of the C57BL/6 mouse strain was examined with a transient transfection assay in which gene expression was monitored by expression of chloramphenicol acetyltransferase. LTRs from Mtv-8 and Mtv-9 were able to direct glucocorticoid-induced chloramphenicol acetyltransferase expression in this assay, while the LTR from Mtv-17 was only about 5% as effective. Analysis of chimeric LTRs indicated that the glucocorticoid-inducible transcriptional enhancer element within the Mtv-17 LTR is active when linked to a functional promoter from Mtv-8, whereas the promoter from Mtv-17 is defective in directing hormone-induced gene expression, even when linked to the Mtv-8 glucocorticoid-responsive enhancer. The DNA sequence of transcriptional control regions of the LTRs of all three endogenous proviral units was determined; this analysis revealed that the source of the defect in Mtv-17 is a single G-to-A transition at position-75 with respect to the site of transcription initiation that resides within the previously defined binding site for the transcription factor nuclear factor 1. Competition experiments with a gel electrophoresis mobility shift assay indicated that the affinity of nuclear factor 1 for DNA derived from Mtv-17 is significantly less than for comparable sequences derived from Mtv-8.

Addition of substitution of simian virus 40 enhancer sequences into the Moloney murine leukemia virus (M-MuLV) long terminal repeat yields infectious M-MuLV with altered biological properties.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-cell lymphoma in mice. The enhancer sequences present within the M-MuLV long terminal repeat (LTR) region of the proviral genome have been shown to influence the disease specificity of the virus strongly. We examined the contribution of the M-MuLV enhancers to the transcriptional activity and pathogenesis of M-MuLV by constructing LTRs containing heterologous enhancer elements. The simian virus 40 enhancer region (72- and 21-base-pair repeats) was inserted into the U3 region (at -150 base pairs) of the M-MuLV LTR (Mo + SV) and also into a deleted form of the LTR which lacks the M-MuLV enhancer sequences (delta Mo + SV). These chimeric LTRs were used to generate infectious M-MuLVs by transfection of corresponding proviral plasmids into mouse fibroblasts. The relative infectivities of Mo + SV and delta Mo + SV recombinant viruses as determined by rat XC cell plaque assay and reverse transcriptase assay were 60 to 70% of wild-type M-MuLV levels. To study the pathogenicity of these two recombinant viruses, we inoculated newborn NIH Swiss mice with either Mo + SV or delta Mo + SV M-MuLV. Both viruses induced disease more slowly than M-MuLV, which induces disease 2 to 4 months postinoculation. Mo + SV M-MuLV-inoculated animals became moribund at 3 to 13 months postinoculation, whereas delta Mo + SV M-MuLV-inoculated animals became moribund at 6 to 24 months postinoculation. The tumors induced by the two viruses were characterized histologically and molecularly. Mo + SV M-MuLV-induced tumors were primarily T-cell-derived lymphoblastic lymphomas containing extensive rearrangements of the T-cell receptor beta gene. In contrast, delta Mo + SV M-MuLV induced pre-B- and B-cell lymphoblastic lymphomas, B-cell-derived follicular-center cell lymphomas, and acute myeloid leukemia. The delta Mo + SV tumor DNAs from B-lineage tumors were typically rearranged at the immunoglobulin gene loci and contained germ line configurations of the T-cell receptor beta gene. Southern blot hybridization confirmed that the tumor DNAs contained the predicted Mo + SV M-MuLV or delta Mo + SV M-MuLV provirus.

Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.

Monocytes were maintained in tissue culture for greater than 3 mo in media supplemented with rCSF-1. These cultures provided susceptible target cells for isolation and propagation of virus from PBMC of HIV-infected patients. HIV isolated into monocytes readily infected other rCSF-1-treated monocytes but only inefficiently infected PHA-stimulated lymphoblasts. Similarly, laboratory HIV strains passaged in T cell lines or virus isolated from patients' leukocytes into PHA-stimulated lymphoblasts inefficiently infected rCSF-1-treated monocytes. Persistent, low-level virion production was detected in macrophage culture fluids by reverse transcriptase activity or HIV antigen capture through 6-7 wk. Marked changes in cell morphology with cell death, syncytia, and giant cell formation were observed in monocyte cultures 2 wk after infection, but at 4-6 wk, all cells appeared morphologically normal. However, the frequency of infected cells in these cultures at 6 wk was 60-90% as quantified by in situ hybridization with HIV RNA probes or by immunofluorescence with AIDS patients' sera. Ultrastructural analysis by EM also showed a high frequency of infected cells; virtually all HIV budded into and accumulated within cytoplasmic vacuoles and virus particles were only infrequently associated with the plasma membrane. Retention of virus within macrophages and the macrophage tropism of HIV variants may explain mechanisms of both virus persistence and dissemination during disease.

Comparative molecular genetic analysis of lymphomas from six inbred mouse strains.

Previous studies of 21 highly lymphomatous AKXD recombinant inbred mouse strains demonstrated correlations between lymphoma type, the somatic proviral DNA content of the lymphoma, and the frequency of virally induced rearrangements in eight common sites of viral integration (Myc, Pim-i, Pvt-1, Mlvi-1, Mlvi-2, Fis-1, Myb, and Evi-1). In this study we analyzed lymphomas from six inbred mouse strains, AKR/J, C58/J, HRS/J (hr/hr and hr/+), SJL/J, SEA/GnJ, and CWD/LeAgl, to determine whether these correlations are also evident in these strains. Mice of the AKR/J, C58/J, and HRS/J strains died exclusively of T-cell lymphomas. In contrast to earlier studies which showed a great disparity in the rate and incidence of lymphomas in HRS/J hr/hr and HRS/J hr/+ mice, we found a high incidence of T-cell lymphomas and the same mean age of onset of disease in both strains. SJL/J mice died primarily of pre-B-cell lymphomas, whereas CWD/LeAgl and SEA/GnJ mice died primarily of B-cell lymphomas. Somatically acquired mink cell focus-forming proviruses were detected only in T-cell lymphomas, whereas ecotropic proviruses were found in lymphomas from all hematopoietic cell lineages. No rearrangements were detected in the Fis-1, Mlvi-2, and Myb loci, whereas rearrangements were detected in the Mlvi-1, Myc, Pim-1, Pvt-1, and Evi-1 loci. Most rearrangements were found in T-cell lymphomas, and many were virally induced. These results are similar to those we obtained previously for lymphomas of 21 highly lymphomatous AKXD recombinant inbred mouse strains.

Inoculation of newborn SWR/J females with an ecotropic murine leukemia virus can produce transgenic mice.

Endogenous ecotropic murine leukemia proviruses that were not present in the parental stock are acquired by the progeny of some SWR/J X RF/J hybrid females. We have made a stock of an ecotropic murine leukemia virus produced by such a hybrid female and inoculated newborn SWR/J females with it. We show that upon crossing of the inoculated females to SWR/J males, some of their progeny acquire ecotropic proviruses. Although most of these proviruses appear to be distributed in somatic tissues in a mosaic way, some are transmitted through the germ line. Thus an exogenous infection is able to mimic the phenomenon observed in SWR/J X RF/J hybrid mice. Available evidence suggests that this infection occurs during oogenesis in the recipient female. Our results document the conversion of an exogenous infectious ecotropic murine leukemia virus to an endogenous provirus without any manipulation of either eggs or embryos.

Integration of bovine leukaemia virus in the ovine genome.

Circulating lymphocytes and tumour cells from 12 sheep experimentally infected with bovine leukaemia virus (BLV), for periods of time varying from 9 to 48 weeks, were analysed for evidence of integrated and unintegrated provirus. Hybridization analysis demonstrated that the provirus was integrated at one or two sites in all cases. Integration was observed at different sites in the animals studied and there was no evidence of unintegrated virus molecules in infected sheep lymphocytes or tumour cells. The data obtained support a monoclonal origin of different tumours in the same sheep.

Hormonal regulation of mouse mammary tumor growth.

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.

[Provirus structure and expression in rat cell lines transformed by the avian sarcoma virus]. / Struktura i ékspressia provirusa v liniiakh kletok krys, transformirovannykh virusom sarkomy ptits.

Two type of exogenous action on transformed cells have been investigated. The first system includes clones of TWERC cells transformed by Rous sarcoma virus and cultivated in the presence of 5-bromodeoxyuridine. The second system consists of spontaneously transformed rat cells Sam IV superinfected with avian sarcoma virus B-77. In these cell lines and clones the structure of the provirus and its expression have been analyzed by different molecular probes.