Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using BspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain. The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant RTs were assayed. The elucidation of the structure of HIV-1 RT makes it possible to determine the locations of specific mutations in the three-dimensional structure of HIV-1 RT [E. Arnold, A. Jacobo-Molina, R. G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature (London) 357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T. A. Steitz, Science 256:1783-1790, 1992]. The mutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase activity without significantly affecting RNase H activity.
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PMID:Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase. 127 5

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
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PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

We have studied the effects of a natural carotenoid, identified as halocynthiaxanthin, on the enzymatic activities associated with the recombinant preparations of the reverse transcriptases (RTs) of human immunodeficiency viruses (HIV) types 1 and 2. The carotenoid was found to be a potent inhibitor of the RNA-dependent DNA polymerase activity (with 50% inhibition obtained at 5-7 microM halocynthiaxanthin), whereas the DNA-dependent DNA polymerase function of both RTs was significantly less sensitive to the inhibitor. Conversely, the ribonuclease H activity associated with the two HIV RTs was essentially insensitive to the carotenoid. The RNA-dependent DNA polymerase function of RT is the only unique activity found in this enzyme that is not expressed at significant levels in uninfected eukaryotic cells. Therefore, it is possible that this carotenoid may serve as a good candidate for the development of novel potent and specific inhibitors of HIV RT.
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PMID:The carotenoid halocynthiaxanthin: a novel inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 137 77

A series of pyrimidine nucleoside analogues containing [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-ribofuranose as the pentose were found to inhibit human immunodeficiency virus type 1 [HIV-1(IIIB)] replication at a concentration of 0.06-0.8 microM but were not cytotoxic at a 1000- to 10,000-fold higher concentration. These nucleoside derivatives were also effective against various other HIV-1 strains, including those resistant to 3'-azido-3'-deoxythymidine, but not against HIV-2, simian immunodeficiency virus, Moloney murine sarcoma virus, or other RNA or DNA viruses. They proved to be highly specific inhibitors of the RNA-dependent DNA polymerase function of the HIV-1 reverse transcriptase, showing no marked inhibition of the HIV-1 reverse transcriptase-associated DNA-dependent DNA polymerase activity, HIV-2 reverse transcriptase, DNA polymerase alpha, herpes simplex virus 1 DNA polymerase, or Thermus aquaticus DNA polymerase.
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PMID:2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2'-dioxide)pyrimidine (TSAO) nucleoside analogues: highlyselective inhibitors of human immunodeficiency virus type 1 that are targeted at the viral reverse transcriptase. 137

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.
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PMID:Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T). 137 14

The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3' termini of the primer DNA was shown to be a major determinant of the infidelity of HIV-1 RT. Human immunodeficiency virus type 2 (HIV-2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA-dependent DNA polymerase activity of HIV-2 RT and compared it with those of RTs of HIV-1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV-2 RT was shown to lack a 3'----5' exonuclease activity. The ability of HIV-2 RT to extend preformed 3'-terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV-2 RT with a specificity of A:C much greater than A:A greater than A:G. The mispair extension appears to be affected mainly by the increase of apparent Km values rather than by the change in Vmax values. The relative extension frequencies from all mispairs with HIV-1 and HIV-2 RTs was 6- to 9-fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error-prone than MLV RT.
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PMID:Fidelity of the reverse transcriptase of human immunodeficiency virus type 2. 137 91

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52

(-)-2'-Deoxy-3'-thiacytidine (3TC) is a selective inhibitor of human immunodeficiency virus replication in vitro (J. A. V. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A. Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron, Antimicrob. Agents Chemother. 36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 +/- 1.0 to 1.24 +/- 5.1 microM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50% inhibited by a 3TC 5'-triphosphate concentration of 23.4 +/- 2.5 microM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50% inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 +/- 31, 24.8 +/- 10.9, and 43.8 +/- 16.4 microM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 microM for DNA polymerase beta and 15.8 +/- 0.8 microM for DNA polymerase gamma.
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PMID:Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma. 138 25

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

We have analyzed the effects of several natural compounds related to avarols and avarones on the catalytic functions of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The most potent substances, designated as avarone A,B and E and avarol F, inhibited indiscriminately the enzymatic activities of HIV-1 RT, namely the RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H. The inhibition of the DNA polymerase activity was found to be non-competitive with respect to either the template-primer or the deoxynucleotidetriphosphate. These studies suggest that the hydroxyl group at the ortho position to the carbonyl group at the quinone ring is involved in blocking the RT activity. The identification of the active site of the inhibitors will hopefully lead to the rational design of new potent anti-HIV drugs.
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PMID:The inhibition of human immunodeficiency virus type 1 reverse transcriptase by avarol and avarone derivatives. 169 11


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