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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To obtain more precise insight into the Mg(2+)-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which is not isomorphous with the Mg(2+)-free crystal previously refined at 1.48 A resolution, was grown at a high MgSO4 concentration more than 100 mM so that even weakly bound Mg2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg(2+)-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 A, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg(2+)-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+ in the metal-bound crystal.
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PMID:Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for a single Mg(2+)-binding site. 810 76

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.
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PMID:Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. 818 16

A 19-nucleotide RNA containing the CUGGGA loop sequence corresponding to nucleotides 30-35 of the HIV-1 trans-activation response element (TAR) was synthesized in vitro and analyzed by biochemical methods and one- and two-dimensional NMR spectroscopy. Diagnostic RNase cleavage patterns were similar for the loops in the full-length HIV-1 TAR and the 19-nucleotide RNA, indicating that they are similar in structure. NMR data showed that the loop is stabilized by base-stacking interactions. The first loop nucleotide is stacked upon the A-helical stem, and the loop uridine is stacked upon this cytosine. On the opposite side of the loop, the third loop guanosine is stacked upon the adenosine, which is stacked upon the stem. No specific Watson-Crick or non-Watson-Crick base pairing across the loop was identified. Unusually short interribose distances indicate a significant distortion of the sugar-phosphate backbone centered at the adenosine. Relatively short NMR relaxation times for protons of the adenosine and its adjacent guanosine, as well as rapidly exchanging imino protons, provide evidence for dynamic processes occurring in the loop.
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PMID:Structural features of an RNA containing the CUGGGA loop of the human immunodeficiency virus type 1 trans-activation response element. 842 39

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.
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PMID:pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells. 865 39

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.
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PMID:Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro. 865 73

Retroviruses contain a dimeric RNA consisting of two identical molecules of genomic RNA. The interaction between the two monomers is thought to occur near their 5'ends. We previously identified a region upstream from the splice donor site, comprising an autocomplementary sequence, responsible for the formation of dimeric HIV-1Lai RNA [Muriaux, D., Girard, P.-M., Bonnet-Mathonire, B., & Paoletti, J.(1995) J. Biol. Chem. 270,8209-8216]. This region appeared to be confined within a putative stem-loop structure. Here we report an in vitro model under conditions of low inioc strength. Two dimers of RNA 77-402 were identified as a function of temperature, and a significant difference was found in their thermostability. Dimer D55, formed at 55 degrees Celsius, is more stable than dimer D37, formed at 37 degrees C. RNase probing experiments confirm the involvement of a stem-loop structure in the dimerization process. In the monomer, the free G257-CGCGC262 sequence forms a loop in the 240-280 region of RNA 77-402, whereas this sequence is engaged in base pairing when D55 and D37 dimers are formed. Our results show that the loop-loop interaction of the autocomplementary G257CGCGC262 sequence, though hydrogen bonding, is responsible for the formation of dimer D37 and strongly suggest that D37 is a "kissing" complex. In contrast, in dimer D55, all the nucleotides of the two hairpin stems, 243-254/264-277, are involved in a complete interstrand interaction.
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PMID:A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro. 866

Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.
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PMID:Inhibition of HIV-1 production and selective degradation of viral RNA by an amphibian ribonuclease. 870 32

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.
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PMID:Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell. 871 Mar 70

We have analyzed by the RNase-A mismatch method 35 isolates from four WHO-sponsored vaccine evaluation sites as a secondary laboratory of the WHO Network for HIV Isolation and Characterization. The application of an estimator for the establishment of genetic distances based on the RNase-A digestion patterns in combination with the phylogenetic analysis has allowed us to construct a tree with five well defined groups of viruses. Because the clustering with known reference strains, samples from Brazil could be grouped as subtype B and the majority of those from Thailand were subtype E. Some of the samples from Uganda were classified as subtype D. Isolates from Rwanda and some from Uganda were identified as subtype A viruses. These results coincide with data obtained by heteroduplex mobility assay and nucleotide sequencing in env regions. The RNase-A mismatch method combined with phylogenetic analysis permitted the primary genetic classification of 33 of 35 samples from the WHO Network.
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PMID:Primary genetic characterization of HIV-1 isolates from WHO-sponsored vaccine evaluation sites by the RNase-A mismatch method. 883 88

The expression of antiviral genes in human hematopoietic stem or progenitor cells has been proposed as a strategy for gene therapy of AIDS. To be successful, this strategy requires safe and efficient transfer of the therapeutic gene into hematopoietic cells and gene expression has to be maintained in HIV susceptible cells following differentiation. We have used retroviral vectors to transfer the gene for a transdominant inhibitor of HIV replication (RevM10) into CD34+ stem/progenitor cells isolated from human umbilical cord blood (UCB). Following transduction, cells were allowed to differentiate either in vitro in clonogenic assays and long-term stromal cell cultures or in human thymus implanted in immunodeficient scid/scid mice in vivo (SCID-hu). Following differentiation and expansion, multiple lineages of cells were shown to carry the transgene. A higher percentage of gene-marked progenitor cells (10-30% in most cases) were detected in methylcellulose colony assays and in long-term stromal cell cultures (1-5%). In contrast, gene-marked T cells derived from transduced CD34+ cells in a SCID-hu model were detected at an even lower frequency (0.01-1%). RevM10 RNA expression was detected in CD34+ cells immediately after transduction and was maintained after in vitro differentiation of those cells into CD14+ myeloid cells. In T cells, the RevM10-specific RNA was detectable by RT-PCR and also by semiquantitative RNase protection. These findings demonstrate that LTR-driven gene expression is sustained in relevant cells derived from retrovirus-transduced hematopoietic progenitor cells after extensive differentiation in vitro and in vivo and suggest that stringent in vivo, rather than in vitro assays, may be a better preclinical system to improve gene marking and expression in hematopoietic cells.
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PMID:Sustained retroviral gene marking and expression in lymphoid and myeloid cells derived from transduced hematopoietic progenitor cells. 885 97


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