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 dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations.
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PMID:Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1. 751 27

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.
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PMID:Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase. 751 79

The E89G alteration in the human immunodeficiency virus type 1 reverse transcriptase has been shown to confer resistance to nucleoside analogs and a loss of magnesium cation preference (Prasad, V.R., Lowy, I., De Los Santos, T., Chiang, L., and Goff, S.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11363-11367. The wild type reverse transcriptase heterodimer, chimeric reverse transcriptases that contain the E89G alteration in one of the subunits (p66wt/p51m and p66m/p51wt), and the mutant enzyme (p66m/p51m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66m/p51m) displayed a higher Vmax, a higher Km for 2'-deoxythymidine triphosphate, and a higher Ki for 2',3'-dideoxythymidine triphosphate than the wild type enzyme. The increased Km and Ki values were observed only when a heterodimer contained the alteration in the p66 subunit. Tests for divalent cation requirement showed that only the dimers containing the wild type p66 (p66wt/p51wt and p66wt/p51m) displayed a preference for magnesium. Our results indicate that p66 plays a dominant role in deoxynucleotide triphosphate substrate recognition (Km), nucleoside analog sensitivity (Ki), and magnesium preference. However, the increased Vmax displayed by the mutant enzyme (p66m/p51m) appeared to be determined by both of the subunits.
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PMID:Subunit-selective mutagenesis of Glu-89 residue in human immunodeficiency virus reverse transcriptase. Contribution of p66 and p51 subunits to nucleoside analog sensitivity, divalent cation preference, and steady state kinetic properties. 751 55

Analysis of the three-dimensional structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with double-stranded DNA indicates that while many nucleoside-resistance mutations are not at the putative dNTP binding site, several are in positions to interact with the template-primer. Wild-type HIV-1 RT and two nucleoside-resistant variants, Leu74-->Val and Glu89-->Gly, have been analyzed to determine the basis of resistance. The ability of the wild-type enzyme to incorporate, or reject, a 2',3'-dideoxynucleoside triphosphate (ddNTP) is strongly affected by interactions that take place between the enzyme and the extended template strand 3-6 nt beyond the polymerase active site. Inspection of a model of the enzyme with an extended template suggests that this interaction involves the fingers subdomain of the p66 subunit in the vicinity of Leu74. These data provide direct evidence that the fingers subdomain of the p66 subunit of HIV-1 RT interacts with the template strand. The wild-type enzyme is resistant to ddITP if the template extension is 3 nt or less and becomes sensitive only when the template extends more than 3 or 4 nt beyond the end of the primer strand. However, the mutant enzymes are resistant with both short and long template extensions. Taken together with the three-dimensional structure of HIV-1 RT in complex with double-stranded DNA, these data suggest that resistance to the dideoxynucleotide inhibitors results from a repositioning or change in the conformation of the template-primer that alters the ability of the enzyme to select or reject an incoming dNTP.
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PMID:Sensitivity of wild-type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug-resistant mutants does not. 751 82

We have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However, protease-defective viruses that contained only p160gag-pol had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3'-azido-3'-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infections for CD4+ cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.
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PMID:DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity. 751 31

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.
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PMID:Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer. 751 28

During DNA synthesis, the binding of human immunodeficiency virus (HIV) reverse transcriptase (RT) to the template-primer precedes its binding to nucleotide triphosphates. The interaction of oligonucleotide DNA with HIV-1 RT was investigated by using a gel retardation assay. Both homodimeric (p66/p66) and heterodimeric (p66/p51) isoforms of HIV-1 RT were capable of binding the DNA oligomers. Thus, all further studies on the interaction of HIV-1 RT with DNA were done with heterodimeric RT. We have studied the conditions for optimal binding. The formation of the RT-DNA complex was primer-independent, and the extent of DNA binding was indistinguishable for both single-stranded and double-stranded DNA (either blunt-ended or recessed). The DNA binding activity of the RT was found to be dependent on oligonucleotide length. HIV-1 RT binds DNA with no apparent sequence specificity. Hence, this enzyme belongs to the sequence nonspecific DNA binding proteins. The interaction was found to be independent of DNA synthesis. The formation of the RT-DNA complex was not influenced by the presence of either template-complementary or noncomplementary dNTPs, indicating that neither DNA polymerization nor binding of the RT to the dNTP affects the stability of the complex. The gel retardation assay was utilized to examine also the effect of various HIV-1 RT inhibitors (i.e., AZT-TP, ddTTP, TIBO, and 3,5,8-trihydroxy-4-quinolone) on the enzyme-DNA interaction. The results indicate differences in the modes of action of these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of the reverse transcriptase of human immunodeficiency virus type 1 with DNA. 752 56

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.
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PMID:High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus. 752 40

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

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
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PMID:Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization. 752 8


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