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PubMed2004

Inhibition of PKR by vaccinia virus: role of the N- and C-terminal domains of E3L.

The process of eukaryotic translation initiation can be regulated by a highly conserved mechanism involving the phosphorylation of the translation initiation factor eIF2 on the alpha subunit. This mechanism is recognized as an efficient step in the host antiviral response. Vaccinia virus (VV), like many other viruses, encodes proteins to overcome this inhibitory process. The C-terminus of the vaccinia virus E3L is known to bind to double-stranded RNA (dsRNA) thereby sequestering the activator of this antiviral response. In this report, the N-terminus of E3L was found to be required for the additional regulation of eIF2alpha phosphorylation. This phosphorylation event did not lead to a global shutdown in protein synthesis. Because the N-terminus of E3L is required for full viral pathogenesis in mice, these results suggest an alternative role of eIF2alpha phosphorylation in regulating viral replication.

Animals↗
PubMed2004

Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3.

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.

Bluetongue virus↗
PubMed2004

Activation of HTLV-I gene transcription by protein tyrosine phosphatase inhibitors.

Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal activation occurred at 8 h and was synergistic with forskolin or PGE(2). Infected cell lines and cells transfected with HTLV-I proviral DNA were equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in this activation.

Cell Line↗
PubMed2005

Activation of human T cell leukemia virus type 1 LTR promoter and cellular promoter elements by T cell receptor signaling and HTLV-1 Tax expression.

Human T cell leukemia virus 1 (HTLV-1) gene expression is regulated by both the viral Tax protein and by cellular transcriptional factors. We have previously shown that immune activation stimuli such as phorbol esters (PMA) and phytohemagglutinin (PHA) cooperate with HTLV-1 Tax expression to enhance HTLV-1 gene expression in infected T cells through increased activity of the HTLV-1 LTR. We now extend these studies to demonstrate roles for the T cell receptor complex, Lck, and Ras molecules in the coactivation of the HTLV-1 LTR by Tax and T cell activation stimuli. We also observe coactivation of Tax-responsive cellular promoter elements containing NF-kappaB and serum response factor (SRF) binding sites by Tax and T cell activation stimuli. These results suggest a model whereby T cell receptor stimulation and Tax expression coactivate HTLV-1 gene expression and cellular gene expression, enhancing activation of latent HTLV-1 and expression of cellular genes involved in disease pathogenesis.

Cell Line↗
PubMed2005

Leukemia virus long terminal repeat activates NFkappaB pathway by a TLR3-dependent mechanism.

The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFkappaB, and because the antiapoptotic and growth promoting activities of NFkappaB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFkappaB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFkappaB signaling pathway via activation of Ras-Raf-IkappaB kinase (IKK) and degradation of IkappaB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR(-/-) mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFkappaB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFkappaB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.

3T3 Cells↗
PubMed2006

Only those genes of the KIAA1245 gene subfamily that contain HERV(K) LTRs in their introns are transcriptionally active.

Insertion of LTRs into some genome locations might seriously affect regulation of the neighboring genes expression. This hypothesis is widely accepted but, however, not confirmed directly. Earlier, we have identified a family of closely related genes highly similar to the KIAA1245 mRNA counterpart. This family included a subfamily of genes some of which contained and the others lacked an LTR in their structure. We compared transcription of several closely related genes of the subfamily differing in the presence or absence of LTRs. Only LTR-containing genes were transcribed in transformed cell lines, tumorous and embryonic human tissues, whereas LTR-lacking genes remained silent. Since the genes were in the same intracellular microenvironment, we suggested that this effect was most probably due to intrinsic cis-characteristics of integrated LTRs and confirmed this by demonstrating high enhancer activity of KIAA1245 LTRs. The expression of the LTR-containing genes in embryonic tissues might suggest their involvement in evolutionary events during primate speciation.

Brain↗
PubMed2006

Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity.

Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30(II)-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30(II)-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30II modulates viral gene expression in a cooperative manner with p300-mediated acetylation.

CREB-Binding Protein↗
PubMed2004

Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

Animals↗
PubMed2006

Rhodopsin-transducin coupling: role of the Galpha C-terminus in nucleotide exchange catalysis.

In the early steps of visual signal transduction, light-activated rhodopsin (R*) catalyzes GDP/GTP exchange in the heterotrimeric G protein (Galphabetagamma) transducin. We recently reported that the catalytic interaction involves two sequential steps. An initial docking between R* and Gbetagamma leads to conformational changes which make the C-terminus of Galpha (CTalpha) available for binding to R*. Binding of CTalpha by R* then triggers GDP/GTP exchange in the Galpha subunit. To further study this two-step mechanism, we investigated different single amino acid substitutions within CTalpha and discuss the effects of high affinity mutations on nucleotide exchange catalysis.

Amino Acid Substitution↗
PubMed2006

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation.

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.

Alleles↗
PubMed2004

Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene.

Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.

Animals↗
PubMed2000

T(-786)--> C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with myocardial infarction, especially without coronary organic stenosis.

Recently, we discovered a T(-786)-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene that is associated with coronary spasm. The precise mechanism(s) of myocardial infarction (MI), especially without coronary organic stenosis, has not been elucidated, but it seems possible that coronary spasm plays a key role in the mechanism. In this study, we examined the frequency with which the T(-786)-->C mutation occurred in 359 patients with MI who were compared with 195 controls. In the MI group, the frequency of C/C, C/T, and T/T genotypes was 1%, 22%, and 77%, respectively. In the control group, the frequency of C/C, C/T, and T/T genotypes were 0%, 8%, and 92%, respectively. The frequency of the C allele was significantly higher in the MI group than in the control group (p < 0.001). In the MI group, 30 of 359 patients (8%) with MI had no stenosed vessels angiographically, 158 (44%) had 1 stenosed vessel, 80 (22%) had 2 stenosed vessels, and 91 (25%) exhibited 3 stenosed vessels. Total and low-density lipoprotein cholesterol levels and the incidence of diabetes mellitus increased as the number of stenosed vessels increased (p < 0.01, respectively). The frequency of the T(-786)-->C mutation was significantly higher in MI patients with no stenosed vessels (50%) than in those with stenosed vessels (p < 0.003). In conclusion, the T(-786)-->C mutation was strongly associated with MI, especially without coronary arterial stenosis, in Japanese patients. The association may be due to the impaired effects of nitric oxide in the cardiovascular system.

Adult↗
PubMed2004

Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically.

Sarcomas can be divided into those with specific translocations displaying monotonous cytomorphology, and those with complex karyotypes and marked cellular pleomorphism. Telomeres contain terminal DNA sequence repeats that maintain chromosomal stability. Telomeres shorten with cell division and may become dysfunctional leading to chromosomal instability. Using a fluorescence in situ hybridization/immunofluorescence method to assess telomere lengths in archival tissues we analyzed these two types of sarcomas using paraffin-embedded primary tumor specimens. Tissues from nine sarcomas with characteristic translocations (two synovial sarcomas, two alveolar rhabdomyosarcomas, two desmoplastic round cell tumors, and one each of infantile fibrosarcoma, myxoid liposarcoma, cellular congenital mesoblastic nephroma) and nine without (four malignant fibrous histiocytomas, two leiomyosarcomas, one pleomorphic rhabdomyosarcoma, one dedifferentiated chondrosarcoma, and one malignant peripheral nerve sheath tumor) were analyzed. In all (nine of nine) cases with specific translocations, which generally have few karyotypic abnormalities, telomere lengths were similar to or reduced compared to surrounding nonneoplastic tissues. In contrast, telomeres in cases lacking specific translocations, which generally contain complex karyotypes, were often found to be dramatically lengthened and heterogeneous. In addition to markedly elongated telomeres, seven of nine (78%) complex cases exhibited large brightly stained regions corresponding to a specific type of promyelocytic leukemia nuclear body found in immortalized cells that maintain telomeres in a telomerase-independent manner [alternative lengthening of telomeres (ALT) pathway]. This phenotype is unlike that of epithelial neoplasms that typically display complex karyotypes with abnormally short telomeres maintained by the enzyme telomerase. The discovery of heterogeneous telomere lengths and evidence of the ALT pathway in the majority of sarcomas with complex karyotypes supports the existence of a telomere maintenance pathway incapable of full karyotypic stabilization in pleomorphic sarcomas. These findings provide additional molecular-genetic evidence supporting the dichotomous grouping of sarcomas into those with characteristic signature translocations without extensive additional karyotypic abnormalities, and those without such signature translocations that typically display very complex karyotypes, and point to telomere dysfunction as a plausible contributor to the chromosomal aberrations found in complex sarcomas.

Cell Nucleus↗
PubMed2000

Retinoic acid stimulates HIV-1 transcription in human neuroblastoma SH-SY5Y cells.

Although the brain is an important target for the human immunodeficiency virus type 1 (HIV) and viral infection causes neuronal degeneration and dementia, the mechanisms responsible for HIV transcription in neuronal cells are largely unknown. We show here that retinoic acid (RA) stimulates HIV transcription in human neuronal SH-SY5Y cells. The steroid receptor coactivator 1 (SRC-1) enhances the transcriptional response to RA, and the viral protein Tat cooperates with RA and SRC-1 to induce a strong transactivation. These results suggest that retinoid receptors could play an important role as activators of viral gene expression in the human brain.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Mutational analysis of the inverted repeats of Tn3.

The transposase protein and the terminal inverted repeat sequences of the prokaryotic transposon Tn3 are essential for transposition. In order to determine the sequences within the inverted repeat necessary for transposition and interaction with transposase, we have constructed a series of mini-Tn3s in which specific mutations have been introduced into the inverted repeats. The effects of these mutations on transposition have been assayed in vivo using a mating-out transposition assay. Several single base-pair mutations within the transposase binding site reduce transposition frequency. Mutations that affect transposition show a greater effect when present in both inverted repeats than when present in only one inverted repeat.

Base Sequence↗
PubMed2003

Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo.

The replication dynamics of simian immunodeficiency virus (SIVmac32H-C8), attenuated through discrete genetic disruption of the nef gene, were compared with the wild-type parental clone (SIVmac32H-J5) using quantitative molecular methods. The primary viraemia of both infections were similar during the first week, but peaked on Day 10 at higher levels for wild-type virus. Viral RNA levels differed most markedly at Day 14. The frequency and levels of viral DNA species, detectable as gag provirus or circular 2-LTR episomes, differed depending on the virus and the lymphoid compartment sampled. 2-LTR circles persisted for prolonged periods in the peripheral blood but were never detected in any SIVmac32H C8-infected tissue, even if positive by gag PCR. Paradoxically, the converse was observed following wild-type infection. 2-LTR circles disappeared from the peripheral blood by Day 42 postinfection but persisted in lymphoid tissues. These findings are discussed in terms of nef and the role and stability of 2-LTR circle forms in vivo.

Animals↗