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.     

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.     

Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences.

Polyomavirus mutants were isolated from PCC4 embryonal carcinoma cells infected with a variant strain of polyomavirus (ev 1001h) and were found to contain a tandem duplication overlapping the enhancers and the origin of replication. These mutants were able to infect several lines of embryonal carcinoma cells, including PCC4, F9, and LT1. The sequence and structure of one of these mutants are presented and compared with those of other PyEC PCC4 mutants previously described.     

Unique long terminal repeat and surface glycoprotein gene sequences of feline leukemia virus as determinants of disease outcome

The outcome of feline leukemia virus (FeLV) infection in nature is variable, including malignant, proliferative, and degenerative disorders. The determinants of disease outcome are not well understood but are thought to include viral, host, and environmental factors. In particular, genetic variations in the FeLV long terminal repeat (LTR) and SU gene have been linked to disease outcome. FeLV-945 was previously identified as a natural isolate predominant in non-T-cell neoplastic and nonneoplastic diseases in a geographic cohort. The FeLV-945 LTR was shown to contain unique repeat elements, including a 21-bp triplication downstream of the enhancer. The FeLV-945 SU gene was shown to encode mutational changes in functional domains of the protein. The present study details the outcomes of infection with recombinant FeLVs in which the LTR and envelope (env) gene of FeLV-945, or the LTR only, was substituted for homologous sequences in a horizontally transmissible prototype isolate, FeLV-A/61E. The results showed that the FeLV-945 LTR determined the kinetics of disease. Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and env gene changed the disease outcome entirely. Further, the impact of FeLV-945 env on the disease outcome was dependent on the route of inoculation. Since the TM genes of FeLV-945 and FeLV-A/61E are nearly identical but the SU genes differ significantly, FeLV-945 SU is implicated in the outcome. These findings identify the FeLV-945 LTR and SU gene as determinants of disease.     

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by cis-platin

We constructed a recombinant plasmid, pBHIV1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the chloramphenicol acetyl transferase (CAT) gene plasmid. Plasmid pBHIV1 also contains the aminoglycoside phosphotransferase gene as a selectable marker. We introduced pBHIV1 in rat 208F fibroblasts and obtained stable geneticin resistant RFBHIV1-1 transfectant cells. A further control used was plasmid p202A, which carries the mutant T24 H-ras1 promoter linked to the promotorless cat gene. Plasmid p202A also carries the aph gene as a selectable marker and was transfected into 208F cells to obtain stable transfectant RF202A-1 cells. Both RFBHIV1-1 and RF202A-1 cells expressed CAT activity from the HIV LTR and T24 H-ras1 promoters. The response to cis-platin, a platin derivative and hexadecyl-phosphocholine was studied on the HIV LTR and H-ras1 regulated CAT activity in RFBHIV1-1 and RF202A-1 cells. It was found that at 5 x 10(-5) M concentrations cis-platin stimulates by 22-fold the expression of CAT from the HIV LTR, whereas only a 4-fold stimulation was observed on the T24 H-ras1 promoter. Our results suggest caution against therapy including this compound at cytotoxic concentrations in the treatment of AIDS patients.     

Deletion of a GC-rich region flanking the enhancer element within the long terminal repeat sequences alters the disease specificity of Moloney murine leukemia virus.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-lymphoblastic lymphoma 2 to 4 months after inoculation. Enhancer sequences in the U3 region of the M-MuLV long terminal repeat, primarily the 75-bp tandem repeats, strongly influence the disease specificity and latency of M-MuLV. We investigated the role of GC-rich sequences downstream of the tandem repeats in the disease specificity of M-MuLV. A recombinant M-MuLV lacking 23 bases of a GC-rich sequence (-174 to -151), Delta 27A M-MuLV, was tested for pathogenesis in neonatal NIH Swiss mice. Delta 27A M-MuLV induced disease with a longer latency than did M-MuLV (7 versus 3 months) in greater than 85% of inoculated mice. More interestingly, this virus showed an expanded repertoire of hematopoietic diseases. Molecular analyses and histopathologic examinations indicated that while 39% of mice inoculated with Delta 27A M-MuLV developed T-cell lymphoblastic lymphoma typical of wild-type M-MuLV, the majority developed acute myeloid leukemia, erythroleukemia, or B-cell lymphoma. Viral DNA corresponding to Delta 27A M-MuLV was detectable in most of the tumors analyzed. These findings indicate that the GC-rich region significantly influences the disease specificity and latency of M-MuLV.     

Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus.

The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.     

Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses.

Molecular clones of the subgroup A feline leukemia virus FeLV-A/Glasgow-1 have been obtained. Nucleotide sequence analysis of the 3' end of the proviral genome and comparison with the published sequence of FeLV-B/Gardner-Arnstein showed that the most extensive differences are located within the 5' domain of the env gene. Within this domain, several divergent regions of env are separated by more conserved segments. The 3' end of env is highly conserved, with only a single amino acid coding difference in p15env. The proviral long terminal repeats are also highly conserved, differing by only eight base substitutions and one base insertion. Specific probes constructed from the FeLV-A or FeLV-B env genes were used to compare the env genes of various exogenous FeLV isolates and the endogenous FeLV-related proviruses of normal cat DNA. An FeLV-A-derived env probe showed no hybridization to normal cat DNA but detected all FeLV-A and FeLV-C isolates tested. In contrast, an FeLV-B env probe detected independent FeLV-B isolates and a family of endogenous FeLV-related proviruses. Our observations provide strong evidence to support the hypothesis that FeLV-B viruses have arisen by recombination between FeLV-A and endogenous proviral elements in cat DNA.     

Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner.

We tested the ability of sequences in the long terminal repeat (LTR) of a mink cell focus-forming (MCF) murine leukemia virus to function as an enhancer in a cell-type-specific manner. In a stable transformation assay, the MCF or Akv LTR and the simian virus 40 enhancer had similar activities in murine fibroblasts. In contrast, the MCF LTR had a significantly greater activity in murine T lymphoid cells than did either the simian virus 40 enhancer or the Akv LTR.     

Retroviral characteristics of the long terminal repeat of murine E.Tn sequences.

E.Tn sequences form a family of long moderately repeated sequences which are abundantly transcribed in the pluripotent cell lineage between day 3.5 and 7.5 of early mouse embryogenesis. The structure of the long terminal repeat (LTR) bordering the E.Tn sequences has been investigated by nucleotide sequencing, primer extension and S1 mapping experiments. This has allowed the identification of U3, R and U5 domains, and of several other structural features all of which are characteristics of retroviral LTRs.     

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.     

Important role of the long terminal repeat of the helper Moloney murine leukemia virus in Abelson virus-induced lymphoma.

The helper virus has been shown to play a critical role in the development of lymphoma induced by the defective Abelson murine leukemia virus (A-MuLV). Indeed, A-MuLV pseudotyped with some viruses, such as the Moloney MuLV, has been shown to be highly lymphogenic, whereas A-MuLV pseudotyped with other viruses, such as the BALB/c endogenous N-tropic MuLV, has been shown to be devoid of lymphogenic potential (N. Rosenberg and D. Baltimore, J. Exp. Med. 147:1126-1141, 1978; C. D. Scher, J. Exp. Med. 147: 1044-1053, 1978). To map the viral DNA sequences encoding the determinant of the lymphogenic potential of Moloney MuLV when complexed with A-MuLV, we constructed chimeric helper viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from Moloney MuLV and from BALB/c endogenous N-tropic MuLV. Chimeric helper MuLVs, recovered after transfection of NIH 3T3 cells were used to rescue A-MuLV, and the pseudotypes were inoculated into newborn NIH Swiss, CD-1, and SWR/J mice to test their lymphogenic potential. We found that a 0.44-kilobase-pair PstI-KpnI long terminal repeat-containing fragment from the Moloney MuLV was sufficient to confer some, but not complete, lymphogenic potential to a chimeric virus (p7M2) in NIH Swiss and SWR/J mice, but not in CD-1 mice. The addition of the 3'-end env sequences (comprising the carboxy terminus of gp70 and all p15E) to the U3 long terminal repeat sequences restored the full lymphogenic potential of the Moloney MuLV. Our data indicate that the 3'-end sequences of the helper Moloney MuLV are somehow involved in the development of lymphoma induced by A-MuLV. The same sequences have previously been found to harbor the determinant of leukemogenicity and of disease specificity of Moloney MuLV when inoculated alone.