Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.     

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.     

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.     

Regions of the Moloney murine leukemia virus genome specifically related to induction of promonocytic tumors.

Moloney murine leukemia virus (MuLV) can be a potent inducer of promonocytic leukemias in mice that are undergoing a chronic inflammatory response. The neoplasms are, at least in part, associated with insertional mutagenesis of the c-myb locus. Evidence is presented for the existence of at least two genetic elements of the virus that are crucial to induction of this disease but are not required for viral replication in hematopoietic tissues or induction of lymphoid disease. These genetic elements were detected by testing the pathogenicity of recombinants between Moloney and Friend MuLVs, the latter of which is nonleukemic to myeloid cells under these conditions, and by testing Moloney MuLV-based viruses that have nonretroviral sequences inserted at specific endonuclease sites in their long terminal repeats (LTRs). Analysis of the Moloney/Friend recombinants showed that there are sequences within the structural gene domain of Moloney, but not Friend, MuLV that are necessary for promonocytic leukemia, whereas the LTRs of the MuLVs are equally effective for promonocytic tumor formation and insertional mutagenesis of the c-myb gene. Experiments with viruses which were mutagenized in the LTR by insertions demonstrated that there is a specific genetic element in the U3 region of the LTR of Moloney MuLV, upstream of the 75-base-pair enhancer which, when interrupted, results in loss of leukemogenicity for cells in the monocytic lineage but not cells in the lymphoid lineage. We conclude, therefore, that promonocytic leukemia induction, in Moloney MuLV-infected mice undergoing a chronic inflammatory response, requires specific sequences in the structural gene region of Moloney MuLV as well as other sequences in the regulatory region of the virus.     

The 3'' region of bovine leukemia virus genome encodes a trans-activator protein.

The genome of bovine leukemia virus (BLV) contains several overlapping, long open reading frames 3' to the envelope gene. Experiments presented here show that the cDNA encompassing the X region open reading frames encodes a trans-activator function capable of increasing the level of gene expression directed by the BLV long terminal repeat sequences. This study provides further evidence of the structural and functional similarities of the bovine leukemia virus and the human T lymphotropic viruses, HTLV-I and HTLV-II.     

Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.     

An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing.

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.