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1.
J Neurovirol ; 7(1): 1-10, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11519477

ABSTRACT

In a substantial number of cases, Human Immunodeficiency Virus type 1 (HIV-1) infection causes neuronal cell loss and leads to the development of AIDS associated dementia. Several studies have suggested that both host and viral factors contribute to neuronal loss. Here we studied the effect of HIV-1 Tat in primary rat neuronal cells as a model to understand mechanism of neuronal cell death. At nano molar concentration, recombinant Tat induced cell death in primary rat mixed cortical neurons. Tat could also induce uptake of calcium in primary rat cultures. When cells were incubated with NMDA receptor antagonists, MK-801 and D-CPP, cell death and 45Ca uptake were inhibited. Under similar conditions non-NMDA antagonists, NBQX, DNQX and CNQX, and sodium channel antagonist, TTX, did not inhibit Tat induced neuronal cell death. In a similar way HIV associated products from in vitro HIV-1 infected cells induced neuronal cell death which was inhibited by NMDA receptor antagonist. Results presented in this paper suggest that activation of NMDA receptors by HIV-1 Tat is responsible for neuronal cell death in primary rat cortical neurons.


Subject(s)
Cerebral Cortex/metabolism , Gene Products, tat/toxicity , HIV-1/metabolism , Neurons/drug effects , Neurons/metabolism , Animals , CD4 Antigens/biosynthesis , Calcium/metabolism , Cell Death/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Culture Media, Conditioned/toxicity , Cytokines/biosynthesis , Dizocilpine Maleate/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/biosynthesis , HeLa Cells/metabolism , HeLa Cells/virology , Humans , Immunosorbent Techniques , Intracellular Fluid/metabolism , Neurons/cytology , Piperazines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Tetrodotoxin/pharmacology , tat Gene Products, Human Immunodeficiency Virus
2.
Antiviral Res ; 49(2): 101-14, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11248362

ABSTRACT

A new class of substituted 2'-benzisothiazolone represented by PD 161374 was discovered with antiviral activity against retroviruses similar to previously described nucleocapsid inhibitor PD 159206 (DIBA-4). In T cell culture, the 50% inhibitory concentrations (EC(50)) of PD 161374 and PD 159206 were on average 2.5 microM (ranges of 1.2-13.5 microM) without any cytotoxic effect up to 100 microM. PD 161374 inhibited acute HIV infection and it was effective when added during the early phase of HIV infection. However, very modest effects were observed in chronically infected H9 cells and the HIV latency model line OM-10.1. Direct PCR analysis of infected cells demonstrated that PD 161374 delayed the appearance of completed HIV-cDNA products including 2LTR circles. Together all these results suggest that PD 161374 exerts its antiviral effect at pre-integration steps in the early phase of the virus life cycle. When combined with a protease inhibitor, PD 161374 did not show any antagonism and combination with a reverse transcriptase inhibitor (AZT) resulted in a synergistic effect.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Thiazoles/pharmacology , Cell Line , Drug Therapy, Combination , HIV Infections/virology , HIV-1/physiology , Humans , Reverse Transcriptase Inhibitors/pharmacology , Thiazoles/chemistry , Virus Replication/drug effects , Zidovudine/pharmacology
3.
Arch Virol ; 146(12): 2385-400, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11811687

ABSTRACT

Intermolecular crosslinking of the retroviral gag structural proteins, capsid (CA) and nucleocapsid (NC), by oxidation of cysteine thiols interferes with virus assembly and infectivity. PD156202 is a dithiobisbenzamide with broad antiviral activity against retroviruses. Treatment of cell free Moloney murine leukemia virus (MuLV) particles with PD156202 induced crosslinking of gag structural proteins and inhibited reverse transcription in mellitin permeabilized virions. Site directed mutagenesis of NC cysteines in the zinc finger of MuLV proviral DNA resulted in virus particles that were noninfectious. The NC mutant virus did not display any intermolecular crosslinking following treatment with PD156202 under nonreducing conditions, which supported that NC cysteine residues participated in PD156202 mediated crosslinking. Replication of MuLV NC mutant virus could be restored by independent expression of wild type NC, making it susceptible to PD156202. These results demonstrate that oxidation of NC cysteines are detrimental for virus assembly and that complementation with wild type NC restored the nucleocapsid protein activity.


Subject(s)
Cross-Linking Reagents/pharmacology , Moloney murine leukemia virus/metabolism , Mutation , Nucleocapsid Proteins , 3T3 Cells , Animals , Antiviral Agents/pharmacology , Benzamides/pharmacology , Capsid/chemistry , Capsid/genetics , Capsid/metabolism , Cysteine/chemistry , Disulfides/pharmacology , Gene Products, gag/chemistry , Gene Products, gag/genetics , Gene Products, gag/metabolism , Genetic Complementation Test , Mice , Moloney murine leukemia virus/genetics , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Retroviridae Infections/virology , Tumor Virus Infections/virology , Virus Assembly , Virus Replication
4.
Cell Immunol ; 189(2): 160-8, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9790730

ABSTRACT

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic HIV-1 strains to enter the host cells. In this study, we aim to better understand the ligand-binding profiles of CCR5 and the chemokine-receptor usage on leukocyte cells. We found that MCP-2 could bind to CCR5 transfectants with high affinity and cross-compete effectively with RANTES, MIP-1alpha, and MIP-1beta. MCP-2 is a true agonist for CCR5, eliciting a robust chemotactic response in CCR5 transfectants similar to that of the three known CCR5 ligands and exhibiting cross-desensitization with RANTES in the Ca2+ flux response. MCP-4 also bound to CCR5 with high affinity and was efficiently displaced by other CCR5 ligands. However, MCP-4 only partially displaced the binding of radiolabeled MIP-1alpha and caused a chemotactic response only at high concentrations. Furthermore, MCP-2 inhibited the binding of the M-tropic HIV-1 gp120 envelope glycoprotein to CCR5 and HIV-1 infection of peripheral blood mononuclear cells. More importantly, we found that MCP-2 could bind and elicit chemotaxis in CD3-activated and IL-2-maintained T cells, and most of these functions could be specifically inhibited by the anti-CCR5 mAb 2D7, whereas the responses mediated by MIP-1alpha or MCP-4 were only partially inhibited by 2D7. Thus, although MCP-2 can bind to and signal through CCR1, CCR2b, and CCR5, among which both CCR2 and CCR5 are expressed at high levels on activated T cells, it appears to preferably utilize CCR5 on these cells. In contrast, MIP-1alpha and MCP-4 seem to activate multiple receptors on the same cells.


Subject(s)
Monocyte Chemoattractant Proteins/metabolism , Receptors, CCR5/metabolism , T-Lymphocytes/metabolism , Animals , COS Cells , Chemokine CCL5/pharmacology , Chemokine CCL8 , Chemotaxis, Leukocyte , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Humans , Lymphocyte Activation , Mice , Monocyte Chemoattractant Proteins/pharmacology , Signal Transduction/drug effects
5.
Biochemistry ; 37(40): 14204-12, 1998 Oct 06.
Article in English | MEDLINE | ID: mdl-9760258

ABSTRACT

TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Long Terminal Repeat/drug effects , HIV-1/genetics , RNA, Viral/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Aminoglycosides , Anti-Bacterial Agents/pharmacology , Anti-HIV Agents/metabolism , Binding, Competitive/drug effects , Binding, Competitive/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Design , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/metabolism , HIV-1/drug effects , HIV-1/physiology , Humans , Quinazolines/pharmacology , Quinoxalines/pharmacology , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Transcriptional Activation/drug effects , Virus Replication/drug effects , Virus Replication/genetics , tat Gene Products, Human Immunodeficiency Virus
6.
Bioorg Med Chem ; 5(6): 1173-84, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9222511

ABSTRACT

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.


Subject(s)
Anti-HIV Agents/pharmacology , Gene Products, tat/drug effects , Gene Products, tat/metabolism , HIV-1/drug effects , HIV-1/physiology , RNA, Viral/antagonists & inhibitors , RNA, Viral/metabolism , Transcription, Genetic/drug effects , Amino Acid Sequence , Aminoglycosides , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Base Sequence , Benzodiazepinones/pharmacology , Camptothecin/pharmacology , HIV-1/metabolism , HeLa Cells/drug effects , Humans , Molecular Sequence Data , Pyrroles/pharmacology , Transcriptional Activation/drug effects , Transcriptional Activation/physiology , Virus Replication/drug effects , Virus Replication/physiology , tat Gene Products, Human Immunodeficiency Virus
7.
Drug Des Discov ; 15(1): 49-61, 1997 May.
Article in English | MEDLINE | ID: mdl-9332831

ABSTRACT

Substituted 2,2'-dithiobisbenzamides and 2-benzisothiazolones were prepared and shown to possess low microM activity with high therapeutic indices against HIV-1, HIV-2 and SIV in cell culture. The mechanism of antiviral action was determined to be directed toward the nucleocapsid protein (NCp7), which contains two zinc fingers and plays vital roles in the viral life cycle. The "active sulfides" of this study cause the extrusion of zinc from these zinc fingers. Structure-activity relationships of the 2,2'-dithiobisbenzamides reveal that the disulfide bond and the ortho benzamide functional groups are essential for activity, with the best compounds having a carboxylic acid, carboxamide, or sulfonamide substituent. The 2-benzisothiazolones are formed from the disulfides both chemically and in vivo and their SAR mimics that of the 2,2'-dithiobisbenzamides. The antiviral activity of the disulfides may require cyclization to the isothiazolones. Two agents, PD 159206 and PD 161374, which showed good antiviral activity, physical properties, and excellent pharmacokinetics in mice, were selected for advanced studies.


Subject(s)
Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Benzamides/chemistry , Benzamides/pharmacokinetics , Thiazoles/chemistry , Thiazoles/pharmacokinetics , Alkylation , Animals , Benzamides/chemical synthesis , Cell Line , Disulfides/chemistry , Disulfides/pharmacology , HIV-1/drug effects , Humans , Mice , Nucleocapsid Proteins/drug effects , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thioamides/chemistry , Thioamides/pharmacology
8.
Proc Natl Acad Sci U S A ; 91(9): 3862-6, 1994 Apr 26.
Article in English | MEDLINE | ID: mdl-8171003

ABSTRACT

We have investigated the molecular basis of human immunodeficiency virus type 1 (HIV-1) latency in a tissue culture model and in HIV-infected people. We show that increased levels of Tat, but not Rev, can release the proviruses from latency in U1 cells. The absence of Tat in these cells is manifested by the accumulation of promoter-proximal viral transcripts, whereas the presence of Tat correlates with increased expression of viral proteins and an increase in promoter-distal transcripts. The presence of promoter-proximal transcripts also serves as a marker for latency in humans. We observed the exclusive presence of promoter-proximal viral transcripts in peripheral mononuclear cells from the majority (10/11) of asymptomatic HIV-infected individuals examined. Activation of these cells in vitro, and viremia in vivo, correlated with a switch from promoter-proximal transcription to promoter-distal transcription. These results suggest that the control between latency and replication of HIV in vivo is at the level of transcription elongation.


Subject(s)
Gene Expression Regulation, Viral , HIV Infections/microbiology , HIV-1/genetics , Virus Latency , Base Sequence , CD4 Antigens/metabolism , Cells, Cultured , DNA Primers/chemistry , Gene Products, rev/genetics , Gene Products, tat/genetics , Genes, rev , Genes, tat , Humans , In Vitro Techniques , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Viral/genetics , Virus Replication , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency Virus
9.
Proc Natl Acad Sci U S A ; 88(18): 8096-100, 1991 Sep 15.
Article in English | MEDLINE | ID: mdl-1896456

ABSTRACT

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs. However, coinjection of Tat and the TAR-containing RNA into oocyte nuclei relieves this translational inhibition [Braddock, M., Chambers, A., Wilson, W., Esnout, M. A., Adams, S.E. & Kingsman, S.M. (1989) Cell 58, 269-279]. We report here that the intramolecular TAR stem-loop structure is a substrate for the double-stranded RNA (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression of HIV-1 is discussed.


Subject(s)
Gene Products, tat/physiology , HIV-1/genetics , RNA, Viral/genetics , Transcriptional Activation , Adenosine , Animals , Base Sequence , Cell Nucleus/physiology , Hydrogen Bonding , In Vitro Techniques , Inosine , Molecular Sequence Data , Molecular Structure , Oocytes , Structure-Activity Relationship , Xenopus laevis , tat Gene Products, Human Immunodeficiency Virus
10.
J Virol ; 64(2): 592-7, 1990 Feb.
Article in English | MEDLINE | ID: mdl-1688626

ABSTRACT

Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA. This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA. To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli. Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H. For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically but at a location 4 bases upstream of the correct site.


Subject(s)
Avian Sarcoma Viruses/genetics , DNA, Viral/genetics , DNA-Directed DNA Polymerase/metabolism , Genes, Viral , RNA, Viral/genetics , RNA-Directed DNA Polymerase/metabolism , Avian Myeloblastosis Virus/enzymology , Base Sequence , Cloning, Molecular , Endoribonucleases/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Leukemia Virus, Murine/enzymology , Molecular Sequence Data , Recombinant Proteins/metabolism , Ribonuclease H
11.
J Virol ; 63(3): 1428-30, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2915386

ABSTRACT

A 179-base fragment of RNA from the 1,679-base antigenome of hepatitis delta virus can not only self-cleave but, when the ends of the resultant fragments are brought into apposition by base pairing to another RNA, also self-ligate. Thus, processing events needed for genome replication in vivo may be strictly RNA mediated.


Subject(s)
Hepatitis Delta Virus/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/genetics , Virus Replication
12.
J Virol ; 62(12): 4439-44, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3184270

ABSTRACT

Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.


Subject(s)
Genes, Viral , Hepatitis D/virology , Hepatitis Delta Virus/genetics , Liver/virology , RNA, Viral/metabolism , Animals , Base Sequence , Cloning, Molecular , Electrophoresis, Agar Gel , Humans , Molecular Sequence Data , Nucleic Acid Hybridization , Pan troglodytes , RNA, Viral/genetics , Templates, Genetic
13.
J Virol ; 62(8): 2674-9, 1988 Aug.
Article in English | MEDLINE | ID: mdl-2455816

ABSTRACT

The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.


Subject(s)
Hepatitis Delta Virus/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , RNA/metabolism , Antigens, Viral/genetics , Base Sequence , Genes, Viral , Hepatitis Delta Virus/immunology , Molecular Sequence Data , Molecular Weight , RNA, Antisense
14.
Nucleic Acids Res ; 15(16): 6705-11, 1987 Aug 25.
Article in English | MEDLINE | ID: mdl-3628005

ABSTRACT

Using SP6 RNA polymerase and various synthetic DNA oligomers as templates (12-65 b), we have obtained efficient synthesis of RNA transcripts without the need for either primer or promoter. Gel analysis of transcripts made from templates of known size demonstrated a predominant single species, together with some minor bands. For a given template the major band had the same 5'-nucleotide as predicted from the 3'-nucleotide of the template. This synthesis procedure makes it possible to efficiently and conveniently make labeled or unlabeled RNA from synthetic DNA oligonucleotides.


Subject(s)
Oligoribonucleotides/chemical synthesis , DNA-Directed RNA Polymerases/metabolism , Oligodeoxyribonucleotides/chemical synthesis , Templates, Genetic , Transcription, Genetic
15.
J Cell Sci Suppl ; 7: 189-95, 1987.
Article in English | MEDLINE | ID: mdl-3503885

ABSTRACT

The retroviral reverse transcriptase is a multifunctional protein. Not only does it contain both RNA- and DNA-directed DNA synthesis activities but also it contains an endonuclease activity necessary for the integration of viral RNA and a RNase H. This latter activity can reduce to oligoribonucleotides viral RNA that has been reverse transcribed into minus-strand DNA. However, during avian retrovirus genome replication it does this in a highly specific manner so as to generate a specific 12-base primer for plus-strand DNA synthesis. Even though many other oligoribonucleotides are also made there is an efficient selection of the specific primer followed by its efficient utilization in plus-strand DNA synthesis, and subsequent removal. We have used a reconstructed system to gain an understanding of the factors that contribute towards these observed specificities.


Subject(s)
DNA Replication , DNA, Viral/genetics , Genes, Viral , Retroviridae/genetics , Virus Replication , Models, Genetic , RNA, Viral
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