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)

Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to beta-D-galactosidase from Escherichia coli in three different ways. Maleimide groups were introduced into beta-D-galactosidase molecules using N,N'-o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into beta-D-galactosidase molecules, which had been treated with excess of 4,4'-dithiodipyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III). Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl-S-acetylmercaptoacetate. The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-beta-D-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N epsilon-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads with (anti-human IgG gamma chain) IgG. The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.
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PMID:Conjugation of recombinant reverse transcriptase of HIV-1 to beta-D-galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG. 751 83

Multiple antigenic peptides (MAP) comprising eight synthetic peptides corresponding to the V3 loop of HIV-1MN or HIV-1IIIB linked to a lysine core were used to characterize the humoral Ab response of HIV-1-infected patients against this domain. One hundred percent of the tested sera from HIV-1-infected European patients reacted significantly with the HIV-1MN V3 loop MAP. Most, but not all, sera also reacted against the HIV-1IIIB V3 loop MAP, albeit with low titers. The in vitro neutralization capacity of the patients' sera against HIV-1MN or HIV-1IIIB infection were tested. All sera showed significant neutralizing titers against HIV-1MN, in comparison with the low (when detectable) neutralizing titers against HIV-1IIIB. Competition assays revealed that the majority of HIV-1MN-neutralizing Abs in patients' sera react with the V3 loop peptides. Only a small fraction of the total neutralizing capacity, presumably specific for other neutralizing epitopes, could not be adsorbed with HIV-1MN V3 loop peptides. The V3 loop peptides could also compete for the binding of Abs in patients' sera mediating Ab-dependent cellular cytotoxicity (ADCC) of HIV-1MN infected cells, inhibiting 30 to 80% of the ADCC activity. Finally, the HIV-1MN V3 loop MAP, when used as immunogen in the presence of RIBI adjuvant without a carrier protein, elicited Abs able to neutralize HIV-1MN (but not HIV-1IIIB) and to initiate ADCC of HIV-1MN-infected cells. This neutralizing and ADCC activity, mediated by V3MN-specific rabbit Abs, could be totally absorbed by V3MN loop peptides.
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PMID:The majority of neutralizing Abs in HIV-1-infected patients recognize linear V3 loop sequences. Studies using HIV-1MN multiple antigenic peptides. 751 20

Determination of the three-dimensional structure of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has indicated a totally different folding for the 51-kDa subunit (p51) than for the 66-kDa subunit (p66). The polymerase catalytic site is located on the p66 subunit. Moreover, the HIV-1-specific RT inhibitors, also designated as the non-nucleoside RT inhibitors (NNRTIs), select for amino acid mutations that afford resistance to these compounds and are clustered in the palm domain of the HIV-1 RT p66 subunit. This pocket is located in the vicinity of, but clearly distinct from, the polymerase active site. However, for the NNRTIs that belong to the class of the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole- 2'',2''-dioxide)] (TSAO) derivatives, the resistance mutation is located at position Glu138. On the p66 subunit, this amino acid is distant from the binding site of the HIV-1-specific RT inhibitors. When the TSAO-specific resistance mutation Glu138-->Lys was introduced solely in the p51 subunit of the RT p66/p51 heterodimer, the enzyme proved completely resistant to TSAO-m3T but retained full sensitivity to TIBO R82150 and ddGTP. On the other hand, when the mutation was introduced only in the p66 subunit the enzyme remained equally sensitive to the inhibitory effects of TSAO-m3T, TIBO R82150, and ddGTP. Our data provide compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the NNRTIs.
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PMID:Resistance of HIV-1 reverse transcriptase against [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2''-dioxide)] (TSAO) derivatives is determined by the mutation Glu138-->Lys on the p51 subunit. 752 83

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-beta-D-galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of epsilon N-2,4-dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound beta-D-galactosidase activity by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers.
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PMID:Diagnosis of HIV-1 infection by detection of antibody IgG to HIV-1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens. 752 37

The human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor quinoxaline S-2720 showed a more-potent inhibitory effect on HIV-1-induced cytopathicity in CEM cells than either nevirapine, pyridinone L-697,661, bis-heteroarylpiperazine (BHAP) U-88204, TSAO ([2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4-amino-1",2"-oxathiole-2",2"-dioxide)-N3-ethylthymine, or 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4-benzodiazepin-2(I H)-one (TIBO) R82913. The quinoxaline derivative was also markedly more inhibitory to the mutant HIV-1 strains containing in their RT Ile-100, Asn-103, Ala-106, Lys-138, Cys-181, or His-188 substitutions than were the other HIV-1-specific RT inhibitors. Moreover, quinoxaline S-2720 totally prevented HIV-1 infection and emergence of drug-resistant mutant virus strains in CEM cell cultures at concentrations (i.e., 0.35 microM) that are 10- to 25-fold lower than those required for BHAP U-88204 and nevirapine to knock out the virus. Also, the concentration-response curve for S-2720 was markedly steeper than for BHAP and nevirapine, as reflected by the ratio of the 95% to the 50% antivirally effective concentration. Lower concentrations of quinoxaline dominantly lead to the appearance of the Ala-106 RT mutation, causing low-level resistance to the compound. At higher quinoxaline concentrations, the Glu-190 RT and/or the Cys-181 RT mutation is added to the Ala-106 mutation, whereas at the highest quinoxaline concentrations, the Ala-106 mutation tends to disappear from the virus pool, leaving the Glu-190 RT and Cys-181 RT mutations as the only mutations conferring high-level resistance to the compound.
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PMID:Resistance pattern of human immunodeficiency virus type 1 reverse transcriptase to quinoxaline S-2720. 752 84

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
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PMID:Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. 752 32

Pyridoxal 5'-phosphate and related compounds were tested for their ability to inhibit gp120-CD4 interaction and human immunodeficiency virus infection in vitro. The results show that pyridoxal 5'-phosphate is a unique CD4 antagonist whose antiviral potency derives from the presence of both lysine-reactive and anionic substituents.
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PMID:Inhibition of gp120-CD4 interaction and human immunodeficiency virus type 1 infection in vitro by pyridoxal 5'-phosphate. 753 Sep 34

To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the National Academy of Sciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV-1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (Ile), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild-type "AZT-sensitive" RTs co-existed with mutated "AZT-resistant" RTs in the same patient sample.
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PMID:Sequence analysis of proviral HIV RT amplified directly by a semi-quantitative technique from AZT treated patients. 753 52

A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the pp6/p51 heterodimer of wild-type RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays.
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PMID:Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3'-dideoxycytidine 5'-triphosphate and other drugs. 753 30

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.
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PMID:Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives). 753 17


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