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

The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases.
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PMID:Fidelity of nucleic acid amplification with avian myeloblastosis virus reverse transcriptase and T7 RNA polymerase. 753 77

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.
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PMID:Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates. 753 96

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.
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PMID:Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. 753 90

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.
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PMID:Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. 754 38

Reverse transcriptase activity is reported in the mononuclear blood cells isolated from a patient in whom paraarticular ossification developed after surgery for an aneurysmal bone cyst. The enzyme was purified to apparent homogeneity by chromatography before being characterized biochemically for its template specificity and ionic requirement. The enzyme was able to transcribe poly(rA).(dT)12-18 very efficiently in the presence of Mn++ ions. Viral particles were observed in the HuT-78 cell line, cocultured with the mononuclear cells of the patient. No viral particles were observed in HuT-78 cells before the coculture. The patient was found seronegative for HIV-1, HIV-2, and HTLV-1. These results suggest that a new retrovirus infecting mononuclear blood cells may be involved in the development of ectopic ossification. This hypothesis is strengthened by the previous finding of a retrovirus in the mononuclear blood cells of a patient with benign osteopetrosis, and by the fact that HTLV-1 infected T-lymphocytes acquire the ability to secrete factors responsible for the lytic bone lesions observed in the patients. A family of human bone diseases that reflect T-cell dysfunction(s) and are caused by lymphotropic viruses may exist.
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PMID:Retroviral expression in a patient who has paraarticular ossification. 754 35

Sequential human immunodeficiency virus type 1 (HIV-1) isolates were obtained over a 29-month period from a person before, during, and after AZT therapy. DNA sequence analysis of polymerase chain-amplified reverse-transcriptase gene showed a gradual accumulation of mutations to peak resistance (IC50 2.13 microM AZT) in association with mutations at codons 44, 210, and 369, as well as at 41, 67, 70, and 215. Eight months after cessation of AZT therapy, when an HIV-1 isolate from the patient was again sensitive to AZT, these mutations had all returned to the pretherapy sequence.
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PMID:Reverse transcriptase mutations in sequential HIV-1 isolates in a patient with AIDS. 756 96

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.
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PMID:The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein. 759 22

Reverse transcriptase (RT) inhibiting antibody in a series of plasma of HIV-1-seropositive subjects was quantitatively measured by poly A-linked colorimetric microtiter plate assay. The plasma were obtained from 6 asymptomatic carrier (AC)s and from 3 patients who progressed to AIDS. They had been followed 29-51 months. RT inhibiting antibody levels in the plasma were measured by inhibition assay against HTLV-IIIB RT activity. In five of the 6 AC cases, RT inhibiting antibodies in the serial plasma maintained high levels, and 50% inhibiting titers of the serial plasma did not decrease throughout the observation periods (45-51 months). HIV isolation from peripheral blood mononuclear cell (PBMC) of these 5 ACs did not succeed, and HIV p24 antigens were not detected in the plasma. In one AC case (046) RT inhibiting antibody levels gradually decreased after 48 months. In this case, HIV p24 antigen was not detected in the serial plasma throughout the observation period (48 months), but HIV was isolated from PBMC after 27 months. On the other hand, RT inhibiting antibody levels in the serial plasma of all 3 patients who progressed to AIDS gradually decreased in observation periods (29-35 months). HIV strains were isolated from these 3 cases. These results suggest that reduction of RT inhibiting antibody levels correlate well with the success of HIV isolation and with progression of clinical manifestation.
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PMID:[Study on reverse transcriptase inhibiting antibody in plasma of HIV-1 seropositive subjects]. 759 75

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.
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PMID:Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter. 761 54

Transcriptional elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex. In prokaryotes its regulation provides an important mechanism of genetic control. Analogous eukaryotic mechanisms are not well understood, but may control expression of proto-oncogenes and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved eukaryotic transcriptional elongation factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through and possibly nascent transcript cleavage. Here we describe the three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human TFIIS. Unlike previously characterized zinc modules, which contain an alpha-helix, this structure consists of a three-stranded beta-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA transactions, including RNAPII itself. This new structure, designated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures and highlights the growing recognition of the beta-sheet as a motif of nucleic-acid recognition.
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PMID:Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS. 762 41


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