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.     

Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3'' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.     

Sodium butyrate activates human immunodeficiency virus long terminal repeat--directed expression

To study the effect of sodium butyrate on human immunodeficiency virus (HIV) long terminal repeat (LTR)--directed expression, we constructed a chimeric plasmid (pLTR-CAT) in which the LTR sequences derived from a molecular clone of HIV were fused to the chloramphenicol acetyltransferase (CAT) gene. We used transient expression assays in transfected tissue culture cells to monitor the activity of the LTR. The expression of the pLTR-CAT plasmid was activated when the cells were exposed to butyrate after transfection. The magnitude of butyrate-induced increase was linear up to an 8 mM concentration and was different with regard to the target promoters used. Recombinant plasmids linked to marker genes may be useful models for studying the effects on HIV of various agents of chemical and biological origin.     

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.     

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.     

trans-Activation of the human T-cell leukemia virus long terminal repeat correlates with expression of the x-lor protein.

Cell lines established directly from adult T-cell leukemia-lymphoma patients or immortalized by human T-cell leukemia virus type I (HTLV-I) in vitro that do not produce complete HTLV virions were characterized both for the content of viral proteins and for the presence of trans-acting factors activating gene expression under the control of the HTLV long terminal repeat. The expression of the 42-kilodalton HTLV x-lor product correlated with trans-activation of the long terminal repeat. The implications of this study for understanding the role of the HTLV x-lor product in the initiation and maintenance of T-lymphocyte transformation are discussed.     

Differential requirements for activation of integrated and transiently transfected human T-cell leukemia virus type 1 long terminal repeat

Adult T-cell leukemia (ATL) cells contain integrated human T-cell leukemia virus type 1 (HTLV-1) proviruses. Although the exact sequence of events leading to the development of ATL remains incompletely resolved, expression of the integrated HTLV-1 long terminal repeat (LTR) is likely required at some point during the process of T-cell transformation. While much has been learned about the regulated expression of transiently transfected LTR reporter plasmids, an analysis of factors required for expression of chromosomally integrated HTLV-1 LTR has not been done. Here, we have constructed CHOK1 and HeLa cells that contain an integrated HTLV-1 LTR-luciferase gene. Using these cells, we have compared the requirements for activation of transiently transfected versus stably integrated HTLV-1 LTR. We observed different requirements for CREB, p300, and P/CAF in the expression of transiently transfected versus stably integrated HTLV-1 LTR. Furthermore, with dominant-negative mutants of CREB, p300, and P/CAF, we found that activation of integrated HTLV-1 LTR by an ambient stress signal, UV-C, proceeds through a path mechanistically distinct from that used by viral oncoprotein, Tax. Our findings point to additional complexities in the regulated expression of HTLV-1 proviruses compared with those hitherto revealed through transfection studies.     

Regulation of the visna virus long terminal repeat in macrophages involves cellular factors that bind sequences containing AP-1 sites.

Visna virus gene expression is highly restricted in monocytes but is induced by monocyte-macrophage differentiation in vivo. Deletion and linker-scanning mutants, gel shift assays, and DNase I footprinting were used to identify sequences in the visna virus long terminal repeat involved in the developmental regulation of gene expression in the U937 monocytic cell line. We found that an AP-1 and an AP-4 binding site were critical for basal activity and that the AP-1 site was required for phorbol ester-inducible gene expression. These results suggest that cellular factors that interact with AP-1 sites are involved in the developmental regulation of visna virus gene expression in macrophages.