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: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of monoclonal antibodies against p51/p66 human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated 1E8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT-primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3'-azido,3'-deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.
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PMID:Monoclonal antibody-mediated inhibition of HIV-1 reverse transcriptase polymerase activity. Interaction with a possible deoxynucleoside triphosphate binding domain. 768 87

We describe in this article some properties concerning the cDNA elongation activity of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT). The kinetic parameters of the polymerization reaction catalyzed by HIV-1 RT, using short templates, were studied. Values of Km and Vmax were measured as a function of the oligoadenylate template length: the logarithm of Km increased linearly, with an incremental factor of 2.2, when the template length differs by one nucleotide. Using short templates, olig(A)n (n = 7-14) and primers shorter or longer than the template, HIV-1 reverse transcriptase was able to synthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as template for cDNA synthesis by RT. In the binding of RT to template of different lengths (5 to 14 nucleotides long), two constants were determined differing in each case by a factor of about 10. The three recombinant forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked to a short template, (pA)14, in the presence of cis-aquahydroxydiamminoplatinum. The efficiency of crosslink of [32P](pA)14 template with each of the subunits of RT correlated well with the affinity of this template to the different forms of RT. In the case of p66/p51, the crosslink occurred mainly with the p66 subunit. These results confirm the important catalytic role of the p66 subunit in the heterodimeric human retroviral polymerase.
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PMID:Human immunodeficiency virus type-1 reverse transcriptase copies very short templates: kinetic and crosslinking analysis. 768 30

Nuclease footprinting has been used to probe features of binary complexes of type 1 human immunodeficiency virus reverse transcriptase (HIV-1 RT) with both natural and synthetic preparations of its cognate replication primer, tRNA(Lys-3). In addition to heterodimeric RT (p66/p51), ribonucleoprotein complexes containing either the p66 or p51 subunit were analyzed. Footprinting experiments employed both structure- and sequence-specific nucleases. Our results indicate a similar mode of interaction for the three RT preparations tested, suggesting contact with each loop of the tRNA primer (D, anticodon, and T psi C), as well as minor perturbation of the anticodon stem. Although there is little evidence for extensive disruption of the 3'-acceptor stem. RNase A footprinting data with natural and synthetic tRNA suggests that potential base pairing between the T psi C and D loops is disrupted in the presence of RT.
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PMID:Nuclease footprinting of human immunodeficiency virus reverse transcriptase/tRNA(Lys-3) complexes. 768 66

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.
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PMID:Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. 768 65

A chromatographic procedure to purify recombinant reverse transcriptase (RT) from human immunodeficiency virus-1 is reported. A bacterial system which expressed large amounts of p66 RT polypeptide was used. The purification scheme was optimized for high-yield production of homogeneous p66/p51 RT using a combination of chromatographic matrices in the following order: Q-Sepharose, heparin-Sepharose, phenyl-Sepharose, S-Sepharose, Poly(A)-Sepharose and Q-Sepharose. The p66 polypeptide remained intact after the first chromatographic step on Q-Sepharose, where it was recovered in the non-adsorbed fraction. A high yield of p66/p51 RT was obtained when the time from application to elution of heparin-Sepharose in the second chromatographic step was prolonged. Phenyl-Sepharose was used in the next chromatographic step to separate the heterodimeric forms of RT from p66 RT on the basis of hydrophobicity. The chromatography on S-Sepharose resolved the major heterodimeric form, p66/p51, from other heterodimeric variants. Further purification was done by affinity chromatography on Poly(A)-Sepharose followed by anion-exchange chromatography on Q-Sepharose. Amounts of 25-35 mg of the pure heterodimer p66/p51 RT were recovered from 50 g of bacterial cells.
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PMID:Increased yield of homogeneous HIV-1 reverse transcriptase (p66/p51) using a slow purification approach. 768 50

Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression vectors. Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits. Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit. This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini. As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- to 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.
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PMID:Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase. 768 66

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic analysis of template.primer interactions with recombinant forms of HIV-1 reverse transcriptase. 769 May 92

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.
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PMID:Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication. 862 54

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.
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PMID:Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization. 867 16

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.
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PMID:dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog. 870 91


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