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

HIV PROTEINASE INHIBITORS: The HIV proteinase enzyme has been identified as a potential target for antiretroviral therapy, as inhibition of this enzyme leads to the generation of immature, non-infectious virions. There are several proteinase inhibitors in development; the first to enter clinical trials was saquinavir. DEVELOPMENT OF SAQUINAVIR: Saquinavir, a transition-stage analogue of an HIV proteinase cleavage site, was developed using computer-led rational design techniques. It is a highly specific inhibitor of HIV-1 and -2 proteinases, with antiviral activity at concentrations 1000-fold less than those causing cytotoxicity. EUROPEAN CLINICAL EXPERIENCE WITH SAQUINAVIR: Three European clinical studies involving 202 patients have been conducted with saquinavir at doses of 25, 75, 200 and 600 mg three times a day. Two studies were dose-ranging monotherapy trials, one in asymptomatic or mildly symptomatic patients not previously treated with zidovudine, the other in patients with advanced HIV infection who had been treated with zidovudine. The third study was a combination therapy trial with zidovudine in previously untreated patients with advanced infection. Saquinavir was well tolerated either alone or in combination with zidovudine. In the monotherapy studies, CD4 cell counts and estimates of viral load showed the best results with the 600-mg dose. The combination of saquinavir and zidovudine resulted in higher and more sustained increases in CD4 cell counts than with either drug alone. The CD4 cell counts favoured saquinavir at 200 and 600 mg in combination with zidovudine, although plasma viraemia and the RNA polymerase chain reaction indicated that the 600-mg dose (in combination) produced better responses.
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PMID:HIV therapy advances. Update on a proteinase inhibitor. 784 Sep 13

Efficient replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) requires the virus transactivator proteins known as Tat. In order to understand the molecular mechanisms involved in Tat transactivation, it is essential to identify the cellular target(s) of the Tat activation domain. Using an in vitro kinase assay, we previously identified a cellular protein kinase activity, Tat-associated kinase (TAK), that specifically binds to the activation domains of Tat proteins. Here it is demonstrated that TAK fulfills the genetic criteria established for a Tat cofactor. TAK binds in vitro to the activation domains of the Tat proteins of HIV-1 and HIV-2 and the distantly related lentivirus equine infectious anemia virus but not to mutant Tat proteins that contain nonfunctional activation domains. In addition, it is shown that TAK is sensitive to dichloro-1-beta-D-ribofuranosylbenzimidazole, a nucleoside analog that inhibits a limited number of kinases and is known to inhibit Tat transactivation in vivo and in vitro. We have further identified an in vitro substrate of TAK, the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation of the carboxyl-terminal domain has been proposed to trigger the transition from initiation to active elongation and also to influence later stages during elongation. Taken together, these results imply that TAK is a very promising candidate for a cellular factor that mediates Tat transactivation.
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PMID:Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. 785 96

Reverse transcriptase-polymerase chain amplification reactions (RT-PCR) were used to identify transcripts for HIV-1 structural and regulatory proteins in peripheral blood mononuclear cells of a cohort of 48 patients. At least one set of PCR primers was capable of detecting HIV-1 transcripts in 94% of patients. Unspliced gag-pol transcripts were detected with gag or pol primer sets in 60 and 63% of samples, respectively. A significant inverse correlation was noted between transcript identification with the gag primer set and the number of CD4-positive lymphocytes in the blood sample and the clinical stage of infection. Single-spliced env transcripts were identified in 44% of individuals. Multiple-spliced tat or nef transcripts were detected in 6.2 and 53% of individuals, respectively. These findings indicate that viral transcripts are expressed throughout the course of HIV-1 infection.
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PMID:Alterations in spliced and unspliced HIV-1-specific RNA detection in peripheral blood mononuclear cells of individuals with varying CD4-positive lymphocyte counts. 790 12

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.
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PMID:Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines. 791 42

The trans-activation response element (TAR) at the 5' end of the human immunodeficiency virus type 1 (HIV-1) mRNAs forms a stable hairpin structure which is a target for binding of the virally encoded protein Tat, which activates viral gene expression, as well as several cellular factors. TAR is also inhibitory to translation. One of several host factors that binds to TAR RNA is the La autoantigen, an RNA-binding protein which functions in RNA polymerase III transcription termination and has also been implicated in cap-independent internal translation initiation on poliovirus RNA. Here we show that La autoantigen alleviates translational repression by the HIV-1 leader RNA. In rabbit reticulocyte lysate, La relieves the cis-inhibitory effect of the TAR RNA on translation of bacterial chloramphenicol acetyltransferase (CAT) mRNA but not inhibition that is mediated by an artificial secondary structure element. Canonical translation factors exhibited slight (eIF-2 and GEF) or no (eIF-4A, eIF-4B, eIF-4E, eIF-4F, eIF-3, and eEF-1 alpha) stimulatory activity on translation of TAR-containing CAT mRNA. In addition, we show that poliovirus RNA, in spite of being an inefficient template in rabbit reticulocyte lysate, is a strong competitive inhibitor of translation of TAR-containing CAT mRNA but not CAT mRNA. This inhibition can be relieved by La but not by any other translation factor. The results suggest a possible involvement of the La autoantigen in HIV-1 gene expression.
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PMID:La autoantigen alleviates translational repression by the 5' leader sequence of the human immunodeficiency virus type 1 mRNA. 793 82

We have examined FIV vif function in the context of molecular clone pF34 (GenBank Accession No. M25729). Rabbit antisera directed against the translation product of the vif gene identified a 29-kDa protein in tissue culture cells infected with FIV-pF34; this protein was not present in cultures of uninfected cells. Thus, the vif gene of this virus strain is expressed in infected cells. Three mutations were made in distinct regions of the vif gene of molecular clone FIV-pF34: (i) deletion of 223 bp from the central portion of the gene, (ii) site-directed mutation of a conserved N-terminal basic region, and (iii) site-directed mutation of a conserved C-terminal motif. FIV proviruses containing each of these mutations were assessed for replication following transfection into two feline adherent cell types, CrFK and G355-5. Reverse transcriptase and p24gag antigen assays of supernatants from transfected cultures revealed that all three vif mutants produced very little cell-free virus or viral protein in both cell types. Results of immunocytochemical staining of these cultures indicated that all three mutants expressed low levels of cell-associated FIV p24gag. These findings suggest that each of the three regions mutated in vif is critical for function. Our observations are consistent with studies showing marked attenuation of HIV-1 vif mutants in certain cell types. We conclude that the vif gene is a critical determinant of FIV-pF34 replication and infectivity in CrFK and G355-5 cells.
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PMID:Analysis of the vif gene of feline immunodeficiency virus. 794 60

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.
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PMID:An efficient expression, purification and immunodetection system for recombinant gene products. 794 24

Nuclear antigen 1 (EBNA-1) is one of the key functions of the oncogenic DNA virus, Epstein-Barr virus (EBV), and is the only viral protein consistently expressed in EBV-associated malignancies. EBNA-1 binds in a site-specific manner to the viral DNA and is essential for viral replication, as well as for maintaining the genome as an extrachromosomal episome within infected cells. EBNA-1 is not recognized by the cellular immune system. Here we demonstrate that, in addition to its known DNA binding properties, EBNA-1 can also act as a strong RNA binding protein, interacting with diverse substrates in vitro, including the EBV-encoded RNA polymerase III transcript EBER1 and the HIV-encoded transactivation response (TAR) element. We also show that EBNA-1 can bind exon sequences derived from its own RNA expressed from the Fp promoter, as found in Burkitt's lymphoma-related cells and in nasopharyngeal carcinomas. EBNA-1 has been identified as a component in an RNA complex; moreover, an anti-EBNA-1 antibody 1H4-1, that does not inhibit DNA binding, blocks binding to RNA. Arginine/glycine-containing (so-called 'RGG') motifs have been found in an increasing number of proteins that interact with RNA. The EBV antigen contains three potential 'RGG' motifs located around an internal glycine/alanine-rich repetitive sequence in the protein, and outside the region of EBNA-1 mapped previously as essential for viral DNA replication and other functionally defined properties. These motifs could be involved in the observed binding between EBNA-1 and RNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:EBNA-1, the major nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding proteins. 795 53

Cationic liposomes can mediate efficient delivery of DNA and DNA/protein complex to mammalian cells in vitro and in vivo. Cationic cholesterol derivatives mixed with phosphatidylethanolamine and sonicated to form small unilamellar vesicles can complex with DNA and mediate the entry into the cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes, is used in a gene therapy clinical trial for melanoma. Recently, we exploited these cationic liposomes for the delivery of trans-activating protein factors to regulate and control the expression of delivered transgenes in a protein dose-dependent manner. Bacteriophage T7 RNA polymerase was co-delivered with a reporter gene under the control of T7 promoter to allow cytoplasmic expression of the gene. Human immunodeficiency virus-1 transactivating protein was also codelivered with a reporter gene under the control of HIV-1 long terminal repeat. Finally, human tumor cells selected for cis-platin resistance or isolated from patients who have failed cis-platin therapy are highly transfectable with cationic liposomes. These results suggest a serial therapy protocol with cis-platin and gene therapy for malignancy.
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PMID:Cationic liposomes for direct gene transfer in therapy of cancer and other diseases. 802 97

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.
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PMID:Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein. 808 57


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