Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.     

Escape from in vivo restriction of Moloney mink cell focus-inducing viruses driven by the Mo+PyF101 long terminal repeat (LTR) by LTR alterations.

Mo+PyF101 M-MuLV is a variant Moloney murine leukemia virus containing polyomavirus F101 enhancers inserted just downstream from the M-MuLV enhancers in the long terminal repeat (LTR). The protein coding sequences for this virus are identical to those of M-MuLV. Mo+PyF101 M-MuLV induces T-cell disease with a much lower incidence and longer latency than wild-type M-MuLV. We have previously shown that Mo+PyF101 M-MuLV is defective in preleukemic events induced by wild-type M-MuLV, including splenic hematopoietic hyperplasia, bone marrow depletion, and generation of recombinant mink cell focus-inducing viruses (MCFs). We also showed that an M-MCF virus driven by the Mo+PyF101 LTR is infectious in vitro but does not propagate in mice. However, in these experiments, when a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF was inoculated into newborn NIH Swiss mice, they died of T-cell leukemia at times almost equivalent to those induced by wild-type M-MuLV. Tumor DNAs from Mo+PyF101 M-MuLV-Mo+PyF101 MCF-inoculated mice were examined by Southern blot analysis. The predominant forms of Mo+PyF101 MCF proviruses in these tumors contained added sequences in the U3 region of the LTR. The U3 regions of representative tumor-derived variant Mo+PyF101 MCFs were cloned by polymerase chain reaction amplification, and sequencing indicated that they had acquired an additional copy of the M-MuLV 75-bp tandem repeat in the enhancer region. NIH 3T3 cell lines infected with altered viruses were obtained from representative Mo+PyF101 M-MuLV-Mo+PyF101 MCF-induced tumors, and mice were inoculated with the recovered viruses. Leukemogenicity was approximately equivalent to that in the original Mo+PyF101 M-MuLV-Mo+PyF101 MCF viral stock. Southern blot analysis on the resulting tumors now predominantly revealed loss of the polyomavirus sequences. These results suggest that the suppressive effects of the PyF101 sequences on M-MuLV-induced disease and potentially on MCF propagation were overcome in two ways: by triplication of the M-MuLV direct repeats and by loss of the polyomavirus sequences.     

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.     

Moloney murine leukemia virus-induced tumors: recombinant proviruses in active chromatin regions.

The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation. DNase I hypersensitive sites were mapped at the junction of cellular sequences and the 5'-viral large terminal repeat (LTR). Expression of a recombinant MuLV seems therefore to be a necessary feature to maintain the transformed state.     

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.     

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.     

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection.The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.