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

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.
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PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers. 137 38

One of the main obstacles for the introduction of PCR method to identify HIV1 proviral DNA in routine diagnostic laboratories is the use of radiolabelled oligodeoxynucleotide probes. Nonradioactive labelled probes have several advantages over radioactive labelling: they are stable for over 1 year, they can be produced easily in large amounts and they are safe. Polymerase chain reaction is an efficient and simple method to produce vector free inserts to use as probes. In this paper we describe a procedure for labelling DNA probes with digoxigenin-11-dUTP using the polymerase chain reaction. This non-radioactive labelling system was applied to detect HIV proviral sequences, amplified in vitro by PCR, from peripheral blood mononuclear cells DNA of infected subjects. We found identical sensitivities and specificities for probes synthesized with the non-radioactive and radioactive labelling procedures. The digoxigenin-11-dUTP can be efficiently incorporated during amplification of a DNA fragment using the polymerase chain reaction. This labelling and detection method proved to be specific, sensible and simple enough to be used in routine diagnostic laboratories for the detection of HIV1 infected individuals.
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PMID:Detection of HIV1 proviral DNA by PCR and hybridization with digoxigenin labelled probes. 152 97

PCR is the most sensitive and direct method for detecting blood-borne viruses, as well as an efficient means for producing vector-free probes. However, the application of PCR, especially in the laboratory diagnosis of human immunodeficiency virus (HIV) infection, is impeded by the current use of radiolabeled oligonucleotide probes. Therefore, we have developed a nonisotopic PCR immunoreactive bead (PCR-IRB) assay to detect HIV type 1 proviral DNA from peripheral blood mononuclear cells (PBMC). We used a biotinylated primer in a set of three oligonucleotides selected from the HIV long terminal repeat region for heminested PCR amplification. An internal probe was synthesized by PCR with incorporation of digoxigenin-labeled dUTP. After solution hybridization of the probe with PCR-amplified products (amplicons), the hybridized DNA was captured with streptavidin-coated magnetic beads. For the detection of hybrids, flow cytometric analyses were carried out by two procedures: (i) direct detection with fluorescein isothiocyanate (FITC)-labeled antidigoxigenin immunoglobulin G (IgG) antibody and (ii) indirect detection with antidigoxigenin sheep IgG antibody followed by FITC-labeled anti-sheep IgG antibody. Both procedures in the PCR-IRB assay detected two to three copies of HIV proviral DNA sequences, a sensitivity that is comparable with that of the conventional radioactive detection of amplicons following probe hybridization and electrophoresis. To compare the PCR-IRB assay with the conventional method, we tested 53 pedigreed PBMC specimens from blood donors and newborns; the results obtained were identical. This nonisotopic PCR-IRB assay can also be automated for potential application in laboratory diagnosis of HIV infection, blood bank screening, and therapeutic monitoring of viremia and perinatal transmission.
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PMID:Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probes. 749 17

An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.
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PMID:Detection of reverse transcriptase activity by enzyme-linked immunosorbent assay in human immunodeficiency virus type 1. 754 28

G-->A hypermutation is a remarkable phenomenon resulting from retroviral reverse transcription in the presence of highly biased dNTP concentrations. Of the three reverse transcriptases (RTases) available, those of human immunodeficiency virus type 1 (HIV-1), avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MoMLV), the HIV-1 enzyme showed the greatest sensitivity to biased [dCTP]/[dTTP] ratios. The HIV-1 RTase was able to discriminate between dUTP, dITP and the four DNA precursors and was insensitive to pH. There was little preference for nucleotide contexts. A few exceptionally modified sequences were found presumably resulting from G-->A hypermutation and multiple strand transfer. This particular predilection of the HIV-1 and, by extrapolation, the lentiviral RTases towards G-->A hypermutation suggests that the phenomenon may have contributed to the remarkably elevated A content of these retroviral genomes.
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PMID:Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction. 754 58

The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.
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PMID:Active site labeling of HIV-1 reverse transcriptase. 768 13

A nonisotopic method for the detection of HIV DNA has been developed, based on the use of avidin-biotin complex to identify the products of polymerase chain reaction (PCR). Biotin label integrated in deoxyuridine triphosphate (bio-11-dUTP) was incorporated in the structure of amplified fragments in the course of PCR. The resultant products were identified in hybridization reaction with specific HIV DNA adsorbed on polystyrene plates. The suggested method was not inferior to the traditional radioisotope method in sensitivity and specificity and helped quantitatively assess the results of experiments using an objective criterion--optic density value.
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PMID:[A nonisotopic method of quantitative PCR analysis in diagnosing HIV infection]. 771 13

A rapid and simple nonradioisotopic method has been developed for detection of polymerase chain reaction (PCR) amplified product. Digoxigenin-11-dUTP (DIG-11-dUTP) was incorporated in the amplified product by including it in the PCR reaction mixture. The PCR product was detected colorimetrically either directly or by reverse phase hybridization method where an unlabelled oligo-nucleotide probe was immobilized on a nitrocellulose dipstick and the digoxigenin labelled PCR product was in the liquid phase. With this system the PCR product could be detected even after 10 cycles of amplification by both direct and hybridization methods. The method was applied on the amplified product of DNA from peripheral blood mononuclear cells from 10 HIV-1 ELISA positive and 8 ELISA negative individuals. PCR was positive in all ELISA positive, Western blot positive individuals from whom HIV-1 was also isolated. PCR was negative in all ELISA negative individuals.
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PMID:A nonradioisotopic reverse phase dipstick hybridization method for detection of polymerase chain reaction amplified product. 775 Oct 42

In the present experiments we have used morphological techniques to study the neuropathological profile of the brain of rats after intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120. Using brain cryostat sections (10 microns) from rats treated with a single, daily dose of gp120 (100 ng/rat) given for 7 and 14 consecutive days, in situ DNA fragmentation was revealed in the neocortex but not in the hippocampus by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL). In these rats, dark degenerating neurones were observed in the neocortex but not in the hippocampus. Treatment with bovine serum albumin (300 ng/rat, i.c.v.) for up to 14 days did not produce DNA fragmentation nor did it yield neuropathological lesions of the neocortex or hippocampus. In conclusion, the present data demonstrate that gp 120 given i.c.v. produced DNA fragmentation in the neocortex, thus suggesting that apoptosis is the mechanism through which neurones of the neocortex are killed.
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PMID:HIV-1 gp120 produces DNA fragmentation in the cerebral cortex of rat. 777 77

We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
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PMID:Detection of HIV-1 DNA by polymerase chain reaction incorporation of digoxigenin-11-dUTP and hybridization to immobilized probes. 796 98


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