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Results for “Terminal Repeat Sequences”

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PubMed1999

No association of HIV type 1 long terminal repeat sequence pattern with long-term nonprogression and in vivo viral replication levels in European subjects.

The HIV-1 long terminal repeat (LTR) promotes and modulates proviral transcription in the infected cell. It has been suggested that truncations and even point mutations in functional sites of the LTR are associated with low viral replication and attenuated pathogenesis in HIV-1-infected long-term nonprogressors (LTNPs). We performed a detailed analysis of LTR sequences from proviral DNA of 21 Italian and Swedish, well-characterized LTNPs and of 15 progressor patients. No truncation was found and no correlation was identified between specific LTR mutations and disease progression. We also failed to find a significant correlation between phylogenetic distance and clinical status. Although HIV-1 LTR interpatient heterogeneity among LTNPs and subjects with HIV-1 RNA levels <500 copies/ml tended to be lower, no sequence mutation was correlated with in vivo viral loads. Our results suggest that HIV-1 LTR defects are rare among Italian and Swedish LTNPs.

Adolescent↗

PubMed1985

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

Here we show that the tissue specificity of murine retrovirus infections is determined by the long terminal repeat (LTR) of an otherwise isogenic set of viruses. The isogenic viruses used for this study contain the coding gag, pol, and env genes of the avirulent Akv virus. Recombinant viruses that contain the LTR of a virus that induces T-cell leukemia lymphoma preferentially infect T lymphocytes. Viruses that carry the LTR of a virus that induces erythroleukemia preferentially infect non-T lymphoblastoid cell lines in the marrow and spleen. The Akv virus itself displays no tissue preference for hematopoietic cells. These experiments suggest that retroviruses that carry appropriate enhancer-promoters can be used to infect selectively specific target cells in animals.

Animals↗

PubMed1984

Generation of AKR mink cell focus-forming viruses: a conserved single-copy xenotrope-like provirus provides recombinant long terminal repeat sequences.

AKV and AKR mink cell focus-forming virus-specific probes from the envelope and long terminal repeat (LTR) regions were prepared for study of the structure of recombinant proviruses in tumor tissues of AKR mice. The results showed that (i) all somatically acquired proviruses possessed, besides a recombinant gp70 gene, an altered U3 LTR; (ii) in a substantial portion of the somatically acquired AKR mink cell focus-forming proviruses, the LTR comprised sequences derived from the same xenotropic-like provirus; (iii) this U3 LTR donating parental provirus (Xeno-dL) was present only once per genome equivalent in several mouse strains; (iv) in the strains containing the Xeno-dL provirus, the provirus was present in the same chromosomal site; (v) restriction analysis of the Xeno-dL revealed that the mink cell focus-forming gp70 sequences were derived from a parental provirus, different from Xeno-dL. Therefore, at least two non-ecotropic parents participate in the generation of leukemogenic AKR mink cell focus-forming viruses: a xenotropic-like virus, Xeno-dL, donating U3 LTR sequences, and another xenotropic-like virus or viruses providing gp70 sequences.

AKR murine leukemia virus↗

PubMed1978

Isolation and characterization of a highly repetitious inverted terminal repeat sequence from Oxytricha macronuclear DNA.

The low-complexity "gene-sized" linear DNA duplexes of the Oxytricha macronucleus sport short inverted terminal repeats; thus, each single strand is capable of forming a circle held together by a duplex "neck" [Wesley, R. D. (1975) Porc. Natl. Acad. Sci. USA 72, 678--682]. We have isolated necks from total, circularized, single-stranded macronuclear DNA by treatment with nuclease S1. Necks represent at least 2.2% of the total DNA, are homogeneous in size (23 base pairs), melt at 55 degrees in 0.18 M Na+, and reassociate extremely rapidly at 22 degrees (Cot1/2 = 1.1 X 10(-5) mol-liter-1.sec) to form hybrid necks of the same thermal stability. From these and other results, we conclude that all necks on all the many thousands of different single-stranded circles are the same. The neck sequence is therefore highly repetitious--found in multiple copies (as inverted terminal repeats at flush duplex ends and probably also internally) on each natural "gene-sized" macronuclear DNA molecule--implying the possible participation of this sequence both in the general vegetative metabolism of macronuclear DNA and in the pre-vegetative process whereby macronuclear DNA is excised from the total Oxytricha genome.

Animals↗

PubMed2000

Monoclonality or oligoclonality of human herpesvirus 8 terminal repeat sequences in Kaposi's sarcoma and other diseases.

BACKGROUND: Infection with human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma (KS)-associated herpesvirus, is associated with all forms of KS, with primary effusion lymphoma (PEL), and with some forms of multicentric Castleman's disease (MCD), but the pathogenic role of HHV8 in these tumors and the clonal nature of KS are still unclear. The purpose of this study was to examine whether the number of terminal repeats (TRs) contained in the fused TR region of HHV8 could be used as a marker of clonality in HHV8-associated tumors. METHODS: Pulsed-field gel electrophoresis (PFGE) and multiple-probe Southern blot analysis of the HHV8 TR region were performed on high-molecular-weight DNA obtained from tumoral KS, PEL, and MCD lesions. RESULTS: These analysis showed that the fused TR region contains a large but variable number of TR units (ranging from 16 to 75) and that the viral genome is present as extrachromosomal circular DNA in these tumors in vivo, with occasional ladders of heterogeneous linear termini reflecting lytic replication. All PEL tumors and PEL-derived cell lines as well as some KS tumors contained monoclonal or oligoclonal fused TR fragments; however, the TR region appeared polyclonal in MCD tumors and in a few KS lesions. CONCLUSION: Several KS and PEL lesions are monoclonal expansions of a single infected cell, suggesting that HHV8 infection precedes tumor growth and thus supporting an etiologic role of latent HHV8 in these proliferations. Our finding that nodular KS lesions display all possible patterns of clonality supports the model according to which KS begins as a polyclonal disease with subsequent evolution to a monoclonal process.

Adult↗

PubMed1998

Neutralizing antibodies against HIV determined by amplification of viral long terminal repeat sequences from cells infected in vitro by nonneutralized virions.

Based on the earliest intracellular synthesis of nascent HIV-1 long terminal repeat (LTR) fragments, we have established a heminested polymerase chain reaction (HNPCR) amplification of the 5' LTR sequences (LTR-HNPCR) for molecular assay of virus-neutralizing antibodies (VNAb). We incubated HIV antibodies with virus isolates for an hour, followed by addition of lymphoid cells (H9 or peripheral blood mononuclear cells [PBMC]) and further incubation for an hour. After washing the cells three times for thorough removal of free virions and antibodies, LTR-HNPCR consistently revealed HIV DNA in H9 cells after 15 minutes, in PBMC after 4 hours, and corresponding virion expression after 7 days in culture. Replication-competent HIV detected by LTR-HNPCR following overnight culture of infected PBMC for 16 to 18 hours was comparable with tissue culture infectivity measured by p24 antigen expression at 7 days. After establishing a molecular assay for in vitro HIV neutralization by HIV Ig, a panel of five HIV isolates tested with 6 monoclonal antibodies and HIV Ig revealed that LTR-HNPCR was comparable with other VNAb assays. These preliminary data indicate that the molecular assay for HIV neutralization has a clear-cut end point, is specific, reliable, and more rapid than other VNAb assays. Therefore, it offers potential utility in evaluating immune response to candidate vaccines.

Cell Line↗

PubMed1991

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.

Animals↗

PubMed1999

Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

Animals↗

PubMed2003

A Moloney murine leukemia virus-based retrovirus with 4070A long terminal repeat sequences induces a high incidence of myeloid as well as lymphoid neoplasms.

Retroviruses can be used to accelerate hematopoietic cancers predisposed to neoplastic disease by prior genetic manipulations such as in transgenic or knockout mice. The virus imparts a second neoplastic "hit," providing evidence that the initial hit is transforming. In the present study, a unique retrovirus was developed that can induce a high incidence of myeloid disease and has a broad host range. This agent is a Moloney murine leukemia virus (Mo-MuLV)-based virus that has most of the U3 region of the long terminal repeat (LTR) replaced with that of retrovirus 4070A. Like Mo-MuLV, this virus, called MOL4070LTR, is NB-tropic and not restricted by Fv1 allelles. MOL4070LTR causes myeloid leukemias in ca. 50% of mice, a finding in contrast to Mo-MuLV, which induces almost exclusively lymphoid disease. The data suggest that the LTR of the 4070A virus expands the tissue tropism of the disease to the myeloid lineage. Interesting, MCF recombinant envelope was expressed in the lymphoid but not the myeloid neoplasms of BALB/c mice. This retrovirus has the potential for accelerating myeloid disease in genetically engineered mice.

Animals↗

PubMed1983

A deletion mutant of mouse mammary tumour virus, lacking 516 nucleotides of the 5' long terminal repeat sequence, can be expressed in a hormone-responsive fashion.

In vitro manipulation of proviral DNA of mouse mammary tumour virus (MMTV) was used to construct mutants with defined deletions at the 5' end of the proviral gene. In the mutants 516, 1400 and 2000 nucleotides were removed from the 5' end. The deleted proviral DNA was tested for transcription and glucorticoid hormone regulation of viral RNA expression upon cotransfection into rat XC tk- cells with a thymidine kinase gene. Intact proviral DNA contained in the plasmid vector pBR322 and the deletion mutant pGR16 delta 516, missing 516 nucleotides of the 5' long terminal repeat (LTR) sequence, were transcribed in a hormone responsive fashion and produced RNA species of 35S and 24S. Deletion of the entire LTR sequence abolished MMTV transcription and the hormonal effect.

DNA, Viral↗

PubMed1997

Genotypic and phenotypic characterization of long terminal repeat sequences from long-term survivors of human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who remain asymptomatic despite prolonged infection present a unique opportunity to understand virologic and immunologic factors involved in the pathogenesis of AIDS. We have previously identified a group of long-term survivors (LTS) who are clinically healthy and immunologically normal despite 13 to 15 years of HIV-1 infection. In this study, we examined the 5' long terminal repeat (5' LTR) sequences in eight of these LTS. A total of 29 nucleotide sequences were obtained from their peripheral blood mononuclear cells (PBMC). Analysis of these sequences revealed no gross deletions within the 5' LTR. Seven of the eight subjects shared nearly identical consensus sequences in the binding sites for NF-kappaB, Sp1, and the viral trans-activator Tat. In multiple samples from one individual (Pt 5), however, G-to-A hypermutations were found throughout the entire region, suggesting a genetically defective 5' LTR. The effects of the observed genetic variations on LTR transcription were studied by transient transfection of an LTR-driven luciferase reporter gene and by infection with a full-length recombinant HIV-1 containing a luciferase reporter (HIVHXBLTRluc). A wide range of basal and Tat-induced transcriptional activities was found among the 5' LTR from seven of the eight LTS in both transfected 293 cells and donor PBMC, suggesting a functionally intact 5' LTR in these individuals. It is therefore unlikely that defects in the 5' LTR are the underlying explanation for the benign clinical course associated with these seven individuals. However, functional abnormalities were found in the LTR from Pt 5 in directing both heterologous and viral gene expression, providing a possible genetic explanation for the low viral load and prolonged asymptomatic state of this individual. Last, a similar overall degree of genetic diversity was found among viruses from the LTS compared to those from patients with AIDS, reinforcing the notion that a strong correlation between the degree of genetic diversity and the rate of disease progression is unlikely.

Base Sequence↗

PubMed2004

Mitotic chromosome-binding activity of latency-associated nuclear antigen 1 is required for DNA replication from terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the persistence of the viral genome during latent infection. It has been suggested that LANA1 tethers the viral genome to the host chromosome and also participates actively in DNA replication from the terminal repeat of KSHV. Here we show by mutational analysis that the mitotic chromosome-binding activity of LANA1 is tightly coupled to its replication activity. Thus, KSHV appears to have evolved a unique tactic for its stable maintenance.

Amino Acid Sequence↗

PubMed1977

Terminally repeated sequences in the avian sarcoma virus RNA genome.

The initiation of DNA synthesis in vitro by RNA-directed DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) of avian oncornaviruses requires a tRNAtrp primer molecule located close to the 5' end of the viral RNA genome. DNA transcripts, 100 nucleotides in length, initiated on the tRNAtrp primer molecule contain nucleotide sequences complementary to a large (25 nucleotides) RNase T1 oligonucleotide, T-13, located at the 5' terminus of the avian sarcoma virus RNA genome. tRNAtrp-initiated DNA transcripts with a length of about 70 nucleotides contain substantially fewer nucleotide sequences complementary to this 5'-terminal oligonucleotide, suggesting that the tRNAtrp primer associated with the avian sarcoma virus RNA is located approximately 100 nucleotides from the 5' end of the RNA. In addition, we present evidence to demonstrate that DNA transcribed from avian sarcoma virus RNA sequences located at the 3' end, immediately adjacent to the poly(A), contains nucleotide sequences that are complementary to the 5'-terminal T1 oligonucleotide T-13. These data indicate that the 5' end of the viral genome contains nucleotide sequences that are repeated at the 3' end of the genome. We conclude that the avian oncornavirus RNA genome is terminally redundant.

Avian Sarcoma Viruses↗

PubMed2004

Accumulation of heterochromatin components on the terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus mediated by the latency-associated nuclear antigen.

In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.

Antigens, Viral↗

PubMed1998

Envelope and long terminal repeat sequences of an infectious murine leukemia virus from a human SCLC cell line: implications for gene transfer.

Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-beta-galactosidase (beta-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter beta-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2.

Amino Acid Sequence↗

PubMed1997

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

DNA, Viral↗

PubMed1991

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjö, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.

Base Sequence↗

PubMed1994

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.

Base Sequence↗