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

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.
 ...
PMID:Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction. 765 9

MAP30 is an anti-HIV plant protein that we have identified and purified to homogeneity from bitter melon (Momordica charantia). It is capable of acting against multiple stages of the viral life cycle, on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase and cell-free ribosome-inactivation activities. We have cloned and expressed the MAP30 gene. The objective of this study is to characterize recombinant MAP30 (re-MAP30) and to determine its anti-HIV, anti-tumor and other activities. We report here that re-MAP30 inhibits HIV-1 and certain human tumors to the same extent as its native counterpart, natural MAP30 (nMAP30). The anti-HIV activity was measured by quantitative focal syncytium formation on CEM-ss cell monolayers, viral core protein p24 expression and viral-associated reverse transcriptase activity in HIV-1-infected H9 cells. The anti-tumor activity was measured by metabolic labeling of protein synthesis in tumor cells. In the dose range of the assay, re-MAP30 exhibits little toxicity to the uninfected viral target cells and other normal human cells. Identical to nMAP30, re-MAP30 is also active in topological inactivation of viral DNA, inhibition of viral DNA integration and cell-free ribosome inactivation. The cloning and expression of the gene encoding biologically active re-MAP30 provides an abundant source of homogeneous material for clinical investigations, as well as structure-function studies of this novel antiviral and anti-tumor agent.
 ...
PMID:Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. 766 70

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.
 ...
PMID:Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase. 766 12

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.
 ...
PMID:Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. 768 10

Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.
 ...
PMID:The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides. 768 36

We have examined components of the preintegration complex of human immunodeficiency virus type 1 (HIV-1) and have analyzed features which govern the association of these components. HIV-1 nucleoprotein complexes, isolated from nuclear and cytoplasmic extracts of CD4+ cells after acute virus infection, contained viral RNA and DNA in association with viral matrix (MA), integrase (IN), and reverse transcriptase (RT) antigens but not capsid (CA) antigens and possessed integration activity in vitro. Association of IN but not RT or MA antigens with viral DNA was detergent-stable. Analysis of viral DNA synthesis and nuclear import of viral nucleoprotein complexes in the presence of a reversible RT inhibitor demonstrated that reverse transcription of viral RNA could be completed entirely in the host cell nucleus. Our studies demonstrate structural and functional features of the nucleoprotein (preintegration) complex of HIV-1 which are pertinent to the understanding of early events in the lentiviral life cycle.
 ...
PMID:Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. 768 60

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
 ...
PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

We present the results from a comparative molecular field analysis (CoMFA) of a set of flavone analogs that inhibit HIV-1 integrase-mediated cleavage (3'-processing step) and integration (strand transfer step) in vitro. The results indicate a strong correlation between the inhibitory activity of these flavones and the steric and electrostatic fields around them. CoMFA quantitative structure-activity relationship models with considerable predictive ability (cross-validated r2 as high as 0.8) were obtained.
 ...
PMID:Three-dimensional quantitative structure-activity relationship (QSAR) of HIV integrase inhibitors: a comparative molecular field analysis (CoMFA) study. 769 4

Certain human immunodeficiency virus type 1 (HIV-1) isolates are able to productively infect nondividing cells of the monocyte/macrophage lineage. We have used a molecular genetic approach to construct two different HIV-1 integrase mutants that were studied in the context of an infectious, macrophage-tropic HIV-1 molecular clone. One mutant, HIV-1 delta D(35)E, containing a 37-residue deletion within the central, catalytic domain of integrase, was noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages. The HIV-1 delta D(35)E mutant, however, exhibited defects in the assembly and/or release of progeny virions in transient transfection assays, as well as defects in entry and/or viral DNA synthesis during the early stages of monocyte-derived macrophage infection. The second mutant, HIV-1D116N/8, containing a single Asp-to-Asn substitution at the invariant Asp-116 residue of integrase, was also noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages but, in contrast to HIV-1 delta D(35)E, was indistinguishable from wild-type virus in reverse transcriptase production. PCR analysis indicated that HIV-1D116N/8 entered monocyte-derived macrophages efficiently and reverse transcribed its RNA but was unable to complete its replication cycle because of a presumed block to integration. These data are consistent with the hypothesis that integration is an obligate step in productive HIV-1 infection of activated peripheral blood mononuclear cells and primary human macrophage cultures.
 ...
PMID:Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1. 770 54

The human immunodeficiency virus type 1 (HIV-1) integrase protein is required for the productive infection of T-lymphoid cells in culture (R. L. LaFemina, C. L. Schneider, H. L. Robbins, P. L. Callahan, K. LeGrow, E. Roth, W. A. Schleif, and E. A. Emini, J. Virol. 66:7414-7419, 1992). This observation suggests that chemical inhibitors of integrase may prevent the spread of HIV in infected individuals. In our search for such potential chemotherapeutic agents, we observed that beta-conidendrol inhibits both the sequence-dependent and sequence-independent endonucleolytic activities of integrase with comparable potencies in vitro (50% inhibitory concentration, 500 nM). Structurally related compounds tested for their abilities to inhibit integrase generated a limited structure-activity analysis which demonstrated that potency is associated with the bis-catechol structure: two pairs of adjacent hydroxyls on separate benzene rings. beta-Conidendrol did not inhibit several other endonucleases and/or phosphoryltransferases. Although beta-conidendrol was not effective in preventing HIV-1 infection in cell culture, the in vitro data demonstrate that it is possible to identify selective agents targeted against this essential HIV-1 function.
 ...
PMID:Inhibition of human immunodeficiency virus integrase by bis-catechols. 772 89


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>