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PubMed1989

trans-activation of viral enhancers including long terminal repeat of the human immunodeficiency virus by the hepatitis B virus X protein.

Human hepatitis B virus contains an open reading frame designated X. We have investigated the trans-activating function of the hepatitis B virus X gene in regulating transcriptional control elements. In the HBV genome the major target for X trans-activation is the enhancer element. Further, the X protein stimulates several other viral promoters/enhancers including the long terminal repeats (LTR) of human retroviruses. One of the viral sequences studied in detail is the human immunodeficiency viral (HIV) LTR which is trans-activated by the X protein. Using mutational analysis of the HIV LTR, we show that the NF-kappa B sequences contained within the U3 region are involved in this stimulatory activity. Nuclear run-on analyses support the notion that X-mediated trans-activation occurs at the level of transcription.

DNA Mutational Analysis↗

PubMed2002

Molecular indications for in vivo integration of the avian leukosis virus, subgroup J-long terminal repeat into the Marek's disease virus in experimentally dually-infected chickens.

Marek's disease virus, a herpesvirus, and avian leukosis virus, subgroup J, a retrovirus, are oncogenic viruses of poultry. Both viruses may infect the same flock, the same bird and the same cell. In a double-infected cell, the retroviral DNA can integrate into the cellular or the Marek's disease virus (MDV) genome. The retroviral-long terminal repeat (LTR) integration into MDV was first described by Isfort et al., (Proc Natl Acad Sci 89, 991-995, 1992) following tissue culture co-infection. The recombinant virus isolated, RM1, had altered biological properties compared to the parental MDV (Witter R.L., Li D., Jones D., and Kung H.-J., Avian Dis 41, 407-421, 1997) . The issue of retroviral sequence integration into herpesviruses in vivo, in cases of double-virus infection is of wide significance in general virology and veterinary medicine; it also represents a special case of gene transposition. Using the avian system, we aimed to determine occurrence of such integrations in vivo. Chickens were experimentally co-infected with both avian leukosis virus (ALV) subgroup J and with MDV. To demonstrate the presence of the retroviral LTR in the MDV genome we applied the Hot Spot-combined PCR assay (Borenshtain R. and Davidson I., J Virol Meth 82, 119-127, 1999) that consisted of two consecutive steps of amplification. By that HS-cPCR assay, certain MDV genomic sites, defined as HS for integration were specifically amplified, the HS step, and then subjected to screening in an attempt to detect LTR inserts. The screening was achieved by amplification using heterologous primer sets, one for the MDV hot spot and the other for the retroviral LTR, the cPCR step. The products were Southern blotted and hybridized with MDV and ALV-LTR probes. Chimeric molecules were detected and evidenced by an intense signal in 3/10 chickens and weakly in other 3/10 birds. Detection was by LTR amplification, sequencing and multiple alignment to the ALV-J-LTR sequence. The present study indicated that chimeric molecules were produced in vivo.

Animals↗

PubMed1994

The long terminal repeats of a murine retrovirus encode a trans-activator for cellular genes.

Type C RNA leukemia viruses cause lymphoid neoplasia after an extended latent period by an unknown mechanism. Infection of human or murine cells with murine leukemia viruses rapidly increases the expression of genes belonging to the immunoglobulin superfamily and involved in T-lymphocyte activation, including the class I major histocompatibility complex antigens. We report here that the long terminal repeat (LTR) of Moloney murine leukemia virus encodes a novel trans-activator, which induces transcription and expression of class I major histocompatibility complex genes. The portion of the LTR responsible for trans-activation lies within the U3 region and can be excised from the LTR while maintaining its activity. Analysis of the U3 region of the LTR and its flanking sequences suggests that functional or regulatory elements for the trans-activity exist between nucleotides 32 and 219 of the LTR sequences. The region of the LTR identified here as important in trans-activation has been shown recently to be a critical determinant of leukemogenicity and latency of Moloney murine leukemia virus. These findings suggest a novel mechanism of retrovirus-induced activation of cellular gene expression, potentially contributing to leukemogenesis.

3T3 Cells↗

PubMed2006

The ribosomal shunt translation strategy of cauliflower mosaic virus has evolved from ancient long terminal repeats.

We have screened portions of the large intergenic region of the Cauliflower mosaic virus (CaMV) genome for promoter activity in baker's yeast (Saccharomyces cerevisiae) and have identified an element that contributes to promoter activity in yeast but has negligible activity in plant cells when expressed in an agroinfiltration assay. A search of the yeast genome sequence revealed that the CaMV element had sequence similarity with the R region of the long terminal repeat (LTR) of the yeast Ty1 retrotransposon, with significant statistical confidence. In plants, the same CaMV sequence has been shown to have an essential role in the ribosomal shunt mechanism of translation, as it forms the base of the right arm of the stem-loop structure that is required for the ribosomal shunt. Since the left arm of the stem-loop structure must represent an imperfect reverse copy of the right arm, we propose that the ribosomal shunt has evolved from a pair of LTRs that have become incorporated end to end into the CaMV genome.

Base Sequence↗

PubMed1990

Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein.

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.

Avian Myeloblastosis Virus↗

PubMed2002

RU5 of Mason-Pfizer monkey virus 5' long terminal repeat enhances cytoplasmic expression of human immunodeficiency virus type 1 gag-pol and nonviral reporter RNA.

Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5' untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5' long terminal repeat (LTR) of Mason-Pfizer monkey virus (MPMV) facilitates Rev/Rev-responsive element-independent expression of HIV-1 gag-pol reporter RNA. The MPMV RU5 region of the LTR is necessary and directs functional interaction with cellular posttranscriptional modulators present in human 293 and monkey COS cells but not in quail QT-6 cells and does not require any viral protein. Deletion of MPMV RU5 decreases the abundance of spliced mRNA but has little effect on cytoplasmic accumulation of unspliced gag-pol RNA despite complete elimination of detectable Gag protein production. MPMV RU5 also exerts a positive effect on the cytoplasmic expression of intronless luc RNA, and ribosomal profile analysis demonstrates that MPMV RU5 directs subcellular localization of the luc transcript to polyribosomes. Our findings have a number of similarities with those of reports on 5' terminal posttranscriptional control elements in spleen necrosis virus and human foamy virus RNA and support the model that divergent retroviruses share 5' terminal RNA elements that interact with host proteins to program retroviral RNA for productive cytoplasmic expression.

5' Untranslated Regions↗

PubMed1990

The product of the c-ets-1 proto-oncogene and the related Ets2 protein act as transcriptional activators of the long terminal repeat of human T cell leukemia virus HTLV-1.

The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Ets1 and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Ets1 and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Ets1 proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5' deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Ets1 was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Tax1 responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition experiments show that integrity of repeats 1 and 4 is important for Ets1 binding. These results show that Ets1 and Ets2 are sequence-specific transcriptional activators. In view of the high level expression of Ets1 in lymphoid cells, Ets1 could be part of the transcription complex which mediates the response to Tax1 and the control of HTLV-1 replication. More generally, Ets1 and Ets2 could regulate transcription of cellular genes.

Animals↗

PubMedDate not supplied

Determination of the leukaemogenicity of a murine retrovirus by sequences within the long terminal repeat.

Although the murine retrovirus SL3-3 is highly leukaemogenic, in both the structure of its genome and in its properties of replication in tissue culture it closely resembles the nonleukaemogenic retrovirus Akv (refs 3, 4). An earlier investigation of the properties of recombinant SL3-3-Akv viruses localized the major determinant of leukaemogenicity outside the env gene, in a region of the viral genome that includes the gag gene and the noncoding long terminal repeat (LTR). To localize the determinant of SL3-3's leukaemogenicity more precisely we have now construced a recombinant provirus containing the LTR of SL3-3 and the coding region of Akv. The leukaemogenicity of these recombinants demonstrates that the determinant of leukaemogenicity lies within the SL3-3 LTR. Nucleotide sequencing of the LTRs of SL3-3 and Akv shows that they differ by a set of changes in the region thought to contain a transcriptional enhancer element. We suggest that enhancer region sequences are the major determinants of leukaemogenicity in these viruses.

Animals↗

PubMed1994

Down-regulation of Rous sarcoma virus long terminal repeat promoter activity by a HeLa cell basic protein.

We have previously isolated a HeLa cell cDNA encoding a 21-kDa polypeptide that is 48% similar to transcription factor IIS. To explore the possibility that p21 plays a role in transcriptional regulation in vivo, we tested the effect of p21 expression on the synthesis of reporter chloramphenicol acetyltransferase (CAT) in transfected COS-1 cells. CAT formation under control of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter was decreased nearly 20-fold in cells coexpressing p21. In contrast, CAT production under control of other sequence elements was only slightly reduced (human immunodeficiency virus type 1 LTR, simian virus 40 early promoter), unaffected (human heat shock protein of 70-kDa promoter, adenovirus major late promoter TATA box), or increased (terminal deoxynucleotidyltransferase initiator element, c-fos promoter) by p21 coexpression as compared to cells cotransfected with the parental vector. The abundance of steady-state CAT transcripts from RSV LTR was also decreased by p21 expression in a dose-dependent manner, suggesting that transcription of RSV LTR/CAT is under negative control by p21. Consistent with an effect on transcription, p21 was localized in nuclei of transfected cells. Deletion analysis of p21 indicated that the sequences essential for inhibition of RSV LTR function include the previously identified ARg/Ser-rich region and zinc finger-like motif. Proliferation of chicken embryo fibroblasts transfected with an infectious molecular clone of RSV was diminished by p21 expression, which also resulted in fewer transformed foci.

Animals↗

PubMed2003

Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells.

Human-tropic porcine endogenous retroviruses (PERV) such as PERV-A and PERV-B can infect human cells and are therefore a potential risk to recipients of xenotransplants. A similar risk is posed by recombinant viruses containing the receptor-binding site of PERV-A and large parts of the genome of the ecotropic PERV-C including its long terminal repeat (LTR). We describe here the unique organization of the PERV-C LTR and its changes during serial passage of recombinant virus in human cells. An increase in virus titer correlated with an increase in LTR length, caused by multiplication of 37-bp repeats containing nuclear factor Y binding sites. Luciferase dual reporter assays revealed a correlation between the number of repeats and the extent of expression. No alterations have been observed in the receptor-binding site, indicating that the increased titer is due to the changes in the LTR. These data indicate that recombinant PERVs generated during infection of human cells can adapt and subsequently replicate with greater efficiency.

Amino Acid Sequence↗

PubMed1987

Human immunodeficiency virus long terminal repeat responds to T-cell activation signals.

Human immunodeficiency virus (HIV), the causative agent of AIDS, infects and kills lymphoid cells bearing the CD4 antigen. In an infected cell, a number of cellular as well as HIV-encoded gene products determine the levels of viral gene expression and HIV replication. Efficient HIV-replication occurs in activated T cells. Utilizing transient expression assays, we show that gene expression directed by the HIV long terminal repeat (LTR) increases in response to T-cell activation signals. The effects of T-cell activation and of the HIV-encoded trans-activator (TAT) are multiplicative. Analysis of mutations and deletions in the HIV LTR reveals that the region responding to T-cell activation signals is located at positions -105 to -80. These sequences are composed of two direct repeats, which are homologous to the core transcriptional enhancer elements in the simian virus 40 genome. Our studies reveal that these elements function as the HIV enhancer. By acting directly on the HIV LTR, T-cell activation may play an important role in HIV gene expression and in the activation of latent HIV.

Base Sequence↗

PubMed1986

Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes.

By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

Animals↗

PubMed1988

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.

Amino Acid Sequence↗

PubMed1988

Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis.

The genomes of human retroviruses [human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus (HTLV-I)] encode positive trans-activator proteins, named tat. In the presence of tat, the transcriptional activity of the homologous HIV-1 or HTLV-I long terminal repeat (LTR) promoter is markedly increased. We have constructed mammalian cell lines that contain stably integrated copies of a HIV-1 or a HTLV-I LTR-chloramphenicol acetyltransferase (CAT) gene. When presynthesized HIV-1 or HTLV-I tat proteins were separately introduced into these cells in the presence of cycloheximide, we found a strong increase in the steady-state expression of the homologous viral LTR. Nuclear "run-on" assays verified that this tat-mediated enhancement, occurring in the absence of de novo cellular protein synthesis, was due to increased transcriptional initiation at the LTR promoter. We conclude that one aspect of transcriptional trans-activation of viral LTR by the HIV-1 and HTLV-I tat proteins does not require the production of new cellular proteins.

Animals↗

PubMed1996

Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site.

Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector.

3T3 Cells↗

PubMed1993

Regulatory elements in the long terminal repeat (LTR) of simian foamy virus type 3 (SFV-3).

Simian foamy virus type 3 (SFV-3) is a retrovirus that has a complex genome organization and encodes two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag, pol, and env. In this report, we demonstrate that ORF-1 of SFV-3 encodes a transcriptional transactivator designated taf (transactivator of foamy virus) which augments gene expression directed by the viral long terminal repeat (LTR). The taf responsive elements have been mapped to the U3 region of the LTR, between positions -637 and -180 (+1 represents the transcription initiation site). Two regions between -637 and -180 in the LTR are targets for taf transactivation. These target sequences for taf confer responsiveness to a heterologous promoter independent of orientation; thus, they function like conditional enhancers. The R-U5 region of the viral LTR is shown to have an inhibitory effect on gene expression. SFV-1 is a related spumavirus and encodes a taf gene that augments expression directed by the SFV-3 LTR as well as the SFV-1 LTR; however, the taf gene of SFV-3 transactivates the SFV-3 LTR but not the SFV-1 LTR. These data on regulatory elements in the SFV-3 LTR show that the mechanism of foamy virus transactivation is significantly different from lentiviruses as well as from the HTLV group of viruses.

Cell Line↗

PubMed1999

Feline leukemia virus long terminal repeat activates collagenase IV gene expression through AP-1.

Leukemia and lymphoma induced by feline leukemia viruses (FeLVs) are the commonest forms of illness in domestic cats. These viruses do not contain oncogenes, and the source of their pathogenic activity is not clearly understood. Mechanisms involving proto-oncogene activation subsequent to proviral integration and/or development of recombinant viruses with enhanced replication properties are thought to play an important role in their disease pathogenesis. In addition, the long terminal repeat (LTR) regions of these viruses have been shown to be important determinants for pathogenicity and tissue specificity, by virtue of their ability to interact with various transcription factors. Previously, we have shown that, in the case of Moloney murine leukemia virus, the U3 region of the LTR independently induces transcriptional activation of specific cellular genes through an LTR-generated RNA transcript (S. Y. Choi and D. V. Faller, J. Biol. Chem. 269:19691-19694, 1994; S.-Y. Choi and D. V. Faller, J. Virol. 69:7054-7060, 1995). In this report, we show that the U3 region of exogenous FeLV LTRs can induce transcription from collagenase IV (matrix metalloproteinase 9) and monocyte chemotactic protein 1 (MCP-1) promoters up to 12-fold. We also show that AP-1 DNA-binding activity and transcriptional activity are strongly induced in cells expressing FeLV LTRs and that LTR-specific RNA transcripts are generated in those cells. Activation of mitogen-activated protein kinase kinases 1 and 2 (MEK1 and -2) by the LTR is an intermediate step in the FeLV LTR-mediated induction of AP-1 activity. These findings thus suggest that the LTRs of FeLVs can independently activate transcription of specific cellular genes. This LTR-mediated cellular gene transactivation may play an important role in tumorigenesis or preleukemic states and may be a generalizable activity of leukemia-inducing retroviruses.

3T3 Cells↗

PubMed1993

Carbon tetrachloride induction of rapid changes in liver nuclear protein factors capable of sequence-specific binding to regulatory elements in the long terminal repeat of polytropic-class endogenous murine leukemia virus-related proviruses.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus-related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCl4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5' of the consensus enhancer sequences) and the 3' terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3-6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCl4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCl4 exposure are discussed.

Animals↗