Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.
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PMID:Lipid- and adenoviral-mediated gene transfer into AIDS-Kaposi's sarcoma cell lines. 962 96

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.
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PMID:A dual-luciferase reporter system for studying recoding signals. 963 Feb 53

A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.
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PMID:Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site. 1057 79

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.
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PMID:Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo. 1113 42

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.
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PMID:Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection. 1114 3

Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3'-P5' phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple helix-forming oligonucleotides (Pso-TFOs) can additionally be photo-induced to become irreversibly bound to their targeted DNA sequence. Here, we have examined the ability of various 15-mer phosphoramidate TFOs targeted to the HIV-1 polypurine tract (PPT) sequence to prevent transcription elongation in cell cultures; the PPT sequence has been cloned in the transcribed region of a reporter firefly luciferase gene (luc) and transient expression experiments performed. We show that the level of transcription inhibition of the reporter gene in cells perfectly correlates with the amount of covalent triplex at the PPT site. The efficacy of non-covalent triplexes (either omitting the irradiation step with the psoralen conjugate, or using the unsubstituted oligonucleotide) has been studied in our expression system; the oligonucleotides were introduced into living cells by cationic lipid-mediated delivery or directly into the cell nucleus by microinjection. This experimental approach allowed us to evaluate the intrinsic activity of triplexes as transcriptional inhibitors; transcription elongation was inhibited in cells in a sequence-dependent and concentration-dependent manner. This experimental system is convenient for quantitative and fast evaluation of new chemistries of antigene oligonucleotides as inhibitors of gene expression in cells and in vivo.
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PMID:Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells. 1117 90

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.
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PMID:Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors. 1150 91

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-residue 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12-mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.
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PMID:Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides. 1172 78

Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.
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PMID:Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1. 1246 32

A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae. Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube. Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein. The assay system has been applied to HIV-1 and L-A directed programmed -1 frameshifting and Ty1 and Ty3 directed +1 frameshifting. The assay system is amenable to high-throughput screening.
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PMID:An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. 1286 12


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