Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The replicative cycle of the human immunodeficiency virus (HIV) is reviewed, and currently used and investigational agents directed against the virus are discussed. The first step in the replication of HIV is selective binding of the envelope glycoprotein to CD4 receptors located on T lymphocytes. The virion is then uncoated within the cytoplasm, yielding viral genomic RNA. Reverse transcriptase uses the viral RNA as a template to form single-stranded DNA, which is duplicated to form proviral DNA through the activity of ribonuclease H. Host RNA polymerases transcribe the integrated proviral DNA into messenger RNA, and there is subsequent translation to viral proteins. After translation, further modification of precursor polyproteins is necessary to produce functional peptides. The assembled virus then buds from the cell surface and invades other cells. Targets of drug intervention in the replicative cycle include (1) binding and entry, (2) reverse transcriptase, (3) transcription and translation, and (4) viral maturation and budding. Inhibitors of binding and entry include recombinant soluble CD4, immunoadhesins, peptide T, and hypericin. Nucleoside reverse-transcriptase inhibitors include zidovudine, didanosine, zalcitabine, and stavudine. Foscarnet, tetrahydroimidazobenzo-diazepinthione compounds, and nevirapine are some nonnucleoside reverse-transcriptase inhibitors. Inhibitors of transcription and translation include antagonists of the tat gene and GLQ223. Castanospermine, N-butyldeoxynojirimycin, and protease inhibitors interfere with viral maturation and budding. Drug combinations that have been or are being investigated include zidovudine plus interferon alfa, zidovudine plus zalcitabine, and zidovudine plus didanosine. Four agents currently have approved labeling for use against HIV infection: zidovudine, didanosine, zalcitabine, and stavudine. Monotherapy with zidovudine remains the treatment of first choice. Although progress has been made in developing drug therapies for HIV infection, more selective and more potent drugs are urgently needed. The best approach at present is to optimize the use of available agents, continue to investigate new therapies, and educate the public about prevention.
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PMID:Agents for treating human immunodeficiency virus infection. 775 75

We report the results of a longitudinal study of RNA splicing patterns in 31 early-stage human immunodeficiency virus disease patients with an average follow-up time of 3 years. Eighteen patients showed no evidence for disease progression, whereas 13 patients either showed a > or = 50% reduction in baseline CD4 count or developed opportunistic infections. Levels of unspliced, tat, rev, and nef mRNAs in peripheral blood mononuclear cells were measured by a reverse transcriptase-quantitative, competitive PCR assay. Viral RNA was detected in all patients at all time points. All 13 rapid progressors had viral RNA loads that were > or = 1 log unit greater than those of the slow progressors. In addition, seven of the rapid progressors showed a reduction of more than threefold in the ratio of spliced to unspliced RNA over the 3 years of follow-up. Conversely, two slow progressors with intermediate levels of viral RNA showed no splicing shift. These results confirm earlier observations that viral RNA is uniformly expressed in early-stage patients. We further show that cellular RNA viral load is predictive of disease progression. Importantly, the shift from a predominately spliced or regulatory viral mRNA pattern to a predominately unspliced pattern both is associated with disease progression and adds predictive utility to measurement of either RNA class alone.
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PMID:Human immunodeficiency virus type 1 cellular RNA load and splicing patterns predict disease progression in a longitudinally studied cohort. 785 28

Antiretroviral drugs already registered or currently in clinical trials are shortly described, including dideoxynucleosides, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, tat inhibitors and antisens molecules. In a second part, the treatment of patients with the nucleosides already on the market or in a pre-registration phase is reviewed, on the basis of an analysis of available phase III clinical trials. The third part describes the strategy of clinical trials and explains why "surrogate markers" including markers of viral replication cannot yet be the main criteria to evaluate the efficacy of a new drug in a phase III trial.
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PMID:[Antiretroviral treatments in human immunodeficiency virus infection]. 793 39

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
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PMID:Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses. 807 30

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.
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PMID:The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. 855 34

To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.
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PMID:Genetic complementation between replication-defective mutants of HIV-1 and SIVagm. 862 49

A new variant of human immunodeficiency virus (HIV)-1 with a mosaic genomic structure was identified in Thailand in 1992. This variant, termed genotype E, was characterized by an envelope gene sequence equidistant from genotypes A through D. The gag gene, encoding the matrix and core virus proteins, grouped with genotype A rather than forming a new clade. More than 500,000 Thais are estimated to be infected with the envelope clade E virus and its type 1 isolates, previously assumed to be rare outliers, are contributing substantially to the global acquired immunodeficiency syndrome pandemic. Reported here is the first complete genomic analysis of one such mosaic virus, isolate CM240 from Thailand. The entire gag-pol region and most of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41. Thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be derived from clade A. Another small segment presumably contributed by the parental E strain has been found in the long terminal repeat. The multiple crossover points detected in CM240 may reflect a common mechanism of frequent strand switching by reverse transcriptase. Full genomic analyses of other mosaic HIV-1 genomes are recommended to determine whether the breakpoints found in CM240 are recurrent and to identify the functional implications of the virion alterations.
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PMID:Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand. 870 15

The authors isolated and characterized a new HIV-1 variant (HIV-1[IbNg]) from the peripheral blood mononuclear cells (PBMCs) of a person living in Nigeria. The virus is highly cytopathic to PBMCs in culture, replicates in primary human T cells and macrophages/monocytes as well as in established human T cell and monocytic cell lines, and it does not induce syncytia in MT-2 cells. Using cytoplasmic RNA from HIV-1[IbNg]-infected PBMCs, five overlapping DNA fragments were amplified through reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into pBluescript II SK(+). DNA sequencing of those fragments indicated that the entire HIV-1[IbNg] genome consists of 9201 nucleotides. Phylogenetic analysis of the variant's env gene sequence showed that the virus clustered with HIV-1 strains belonging to HIV-1 clade A. The following genetic features are unique to this virus: a 16-bp insert in the primer-binding site, a large Rev open reading frame, a Rev-responsive element which is predicted to form a different secondary structure than described for clade B viruses, the potential to encode a heavily glycosylated Env protein, and a frameshift resulting in a stop codon in the tat gene.
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PMID:Genomic structure and nucleotide sequence analysis of a new HIV type 1 subtype A strain from Nigeria. 889 49

Moloney murine leukemia virus (MMLV)-derived pUCMoTiN-based retroviral vectors were engineered to allow constitutive and Tat (trans-activator of transcription)-inducible expression of five hammerhead ribozymes targeted against highly conserved sequences within the group antigen (Gag), protease (Pro), reverse transcriptase (RT), tat, and envelope (Env) coding regions of human immunodeficiency virus type-1 (HIV-1) RNA. Amphotropic retroviral vector particles were used to infect a human CD4+ lymphocyte-derived MT4 cell line. The pool of stable MT4 transductants expressing these ribozymes were each tested for their susceptibility to HIV-1 infection. RzTat conferred no protection to MT4 cells. RZGag and RzRT completely inhibited virus multiplication for 6 days. RzPro and RzEnv conferred the best protection, as they completely inhibited virus production for 12 and 15 days, respectively. No correlation was found between the degree of HIV-1 resistance conferred and the ability of these ribozymes to cleave their target RNA in vitro. From RzPro-expressing HIV-1-infected cells following virus escape, RzPro and target RNA sequences were amplified and checked for cleavage in vitro. The ribozyme expressed in these cells was shown to cleave the corresponding target RNA. Thus, a mutation in the ribozyme or target RNA does not seem to be the mechanism underlying virus escape.
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PMID:Comparative analysis of five highly conserved target sites within the HIV-1 RNA for their susceptibility to hammerhead ribozyme-mediated cleavage in vitro and in vivo. 891 8

An antiidiotypic/clonotypic marker, designated 1F7, is restricted to antibodies directed to human immunodeficiency virus type 1 (HIV-1) envelope, core, and reverse transcriptase proteins. 1F7-Id is shared by more than 70% of HIV-infected individuals, arising early and persisting throughout all disease stages. To study the specificity and biological function of this cross-reactive idiotypic marker, and to explore its potential in therapeutics, we have sought an appropriate animal model. 1F7-Id+ antiviral antibodies are found among experimentally HIV-1 IIIB-infected chimpanzees; however, these rare and expensive animals do not develop acquired immunodeficiency syndrome (AIDS), and so are not an appropriate model. We now report the presence in rhesus monkeys of 1F7-Id on antibodies to the external envelope glycoproteins of simian immunodeficiency virus (SIV). This may be surprising, in view of the genetic and serologic differences between SIV and HIV-1, but is in accord with the occurrence of 1F7-Id on antibodies reacting with a broad range of HIV-1 strains. We also found 1F7-Id on anti-gp120 antibodies in rhesus monkeys infected with chimeric immunodeficiency viruses (SHIV) expressing the env, tat, and rev genes of HIV-1 on a backbone of SIV. Both SIV and SHIV cause AIDS in these monkeys. Thus, SIV- and SHIV-infected rhesus monkeys are suitable models for exploring the role of 1F7 in AIDS pathogenesis and prevention. Experiments are underway using MAb 1F7 to test the hypothesis of deceptive imprinting in AIDS.
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PMID:An HIV-1 infection-related idiotype/clonotype (1F7) is expressed on antibodies directed to envelope glycoprotein in simian immunodeficiency virus- and chimeric simian/human immunodeficiency virus-infected rhesus monkeys. 908 23


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