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 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

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

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

New base-substituted analogs of dCTP containing an azido group have been synthesized and applied to a selective photoaffinity modification of HIV-RT (p66/p51 heterodimer). The labeling of only the 66 kDa subunit of HIV-RT was detected when the enzyme was first irradiated with the analogs and then template (5'-(d)GGTTAAATAAAATAGTAAGAATGTATAGCCCCTACCA-3') and 5' 32P end-labeled 3'-(d)TTACATATCGGGGATGGT-5' primer were added. The 5' 32P end-labeled primer elongated by dCTP analogs in the presence of both HIV-RT and DNA template is able to modify both subunits of HIV-RT and DNA template. This way of specific cross-linking to both DNA (RNA) template and HIV-RT opens up new possibilities to study the HIV-RT active site.
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PMID:Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs. 752 54

The locations of HIV-1 RT nucleoside and non-nucleoside inhibitor-binding sites and inhibitor-resistance mutations are analyzed in the context of the three-dimensional structure of the enzyme and implications for mechanisms of drug inhibition and resistance are discussed. In order to help identify residues that may play a role in inhibitor binding, solvent accessibilities of amino acids that comprise the inhibitor-binding sites in the structure of HIV-1 RT complexed with a dsDNA template-primer are analyzed. While some mutations that cause resistance to nucleoside analogs, such as AZT, ddI, and ddC, are located near enough to the dNTP-binding site to directly interfere with binding of nucleoside analogs, many are located away from the dNTP-binding site and more likely confer resistance by other mechanisms. Many of the latter mutations are located on the surface of the DNA-binding cleft and may lead to altered template-primer positioning or conformation, causing a distortion of the geometry of the polymerase active site and consequent discrimination between normal and altered dNTP substrates. Other nucleoside analog-resistance mutations located on the periphery of the dNTP-binding site may exert their effects via altered interactions with dNTP-binding site residues. The structure of the hydrophobic region in HIV-1 RT that binds non-nucleoside inhibitors, for example, nevirapine and TIBO, has been analyzed in the absence of bound ligand. The pocket that is present when non-nucleoside inhibitors are bound is not observed in the inhibitor-free structure of HIV-1 RT with dsDNA. In particular it is filled by Tyr181 and Tyr188, suggesting that the pocket is formed primarily by rotation of these large aromatic side-chains. Existing biochemical data, taken together with the three-dimensional structure of HIV-1 RT, makes it possible to propose potential mechanisms of inhibition by non-nucleoside inhibitors. One such mechanism is local distortion of HIV-1 RT structural elements thought to participate in catalysis: the beta 9-beta 10 hairpin (which contains polymerase active site residues) and the beta 12-beta 13 hairpin ("primer grip"). An alternative possibility is restricted mobility of the p66 thumb subdomain, which is supported by the observation that structural elements of the non-nucleoside inhibitor-binding pocket may act as a "hinge" for the thumb.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. 752 66

Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding.
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PMID:The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases. 752 38

We constructed plasmid vectors that simultaneously express both the p66 and p51 subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in Escherichia coli. These vectors allow us to generate HIV-1 RT heterodimers in which either the p66 or the p51 subunit has the wild-type sequence and the other subunit has a specific amino acid substitution. We used these vectors to express HIV-1 RT heterodimers containing several different amino acid substitutions reported to confer resistance to nonnucleoside inhibitors. Most of the amino acid substitutions conferred resistance to nonnucleoside inhibitors R86183 (TIBO) and TSAO-m3T only when present in the p66 subunit of the p66-p51 heterodimer; heterodimers that contained a wild-type p66 subunit and a mutant p51 subunit remained sensitive to the inhibitors. However, there was one mutation, E138K, that conferred drug resistance when the mutation was present in the p51 subunit. The corresponding heterodimer with the E138K mutation in the p66 subunit and a wild-type p51 subunit remained sensitive to the inhibitors. Analysis of the three-dimensional structure of HIV-1 RT indicated that residue 138 of the p51 subunit is in the nonnucleoside inhibitor-binding pocket while residue 138 of the p66 subunit is not. The mutagenesis results, combined with structural data, support the idea that the nonnucleoside inhibitors exert their effects by binding to a hydrophobic pocket in the RT heterodimer and that mutations which give rise to drug resistance directly interfere with the interactions between the nonnucleoside inhibitors and HIV-1 RT.
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PMID:Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase. 752 11

The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.
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PMID:Analysis of HIV type 1 reverse transcriptase expression in a human cell line. 753 25

In the interaction between HIV-1 RT and tRNA(Lys3) each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.
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PMID:Interaction of primer tRNA(Lys3) with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation. 753 48


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