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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We produced a series of monoclonal antibodies against the human immunodeficiency virus (HIV-1) reverse transcriptase by immunizing mice with either purified recombinant HIV-1 p66 protein or with recombinant vaccinia virus which expresses HIV-1 pol sequences. The antibodies generated were specific for the reverse transcriptase protein, and recognized only the p51 and p66 subunits of the enzyme in each of the HIV-1 viral lysates and lysates of HIV-1 infected cells. The antibodies did not cross-react with HIV-2 reverse transcriptase. Most important, several of the antibodies are unique, in that they are the first that can bind to sites close to the N-terminal. This latter region has been suggested to form part of the polymerase domain of the reverse transcriptase. None of the antibodies could neutralize either the RNA-dependent DNA polymerase or RNase H activities of either p66 or p51/66 proteins. The binding patterns of these various antibodies to p66 and p51/66 were dependent on each of three independent variables: the source of antigen amployed, the individual specificity of the antibody, and the method employed to detect reactivity. These monoclonal antibodies provide useful reagents for the study of reverse transcriptase native structure-function relationships.
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PMID:Generation and characterization of murine monoclonal antibodies reactive against N-terminal and other regions of HIV-1 reverse transcriptase. 768 57

The human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors [tetrahydroimidazo[4,5,1-jk] [1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine, nevirapine, pyridinone, bis(heteroaryl)piperazine, etc.] are potent inhibitors of HIV-1 replication in cell culture. The rapid emergence of drug-resistant escape mutants in vitro (cell culture) and in vivo (patients) is predominantly linked to the Y181C mutation. Because amino acids Y181 and Y188 appear to be located within the drug binding site of the enzyme, we studied the impact of mutations of both amino acids on the enzyme kinetics and on the susceptibility of the enzyme to different HIV-1-specific RT inhibitors. Mutations Y181C, Y181I, and Y188L led to reduced sensitivity, albeit of varying extents, to all HIV-1-specific RT inhibitors. No resistance was observed to 2',3'-dideoxyguanosine 5'-triphosphate or phosphonoformic acid. The kcat of the Y181C mutant was similar to that of the wild-type RT (18 sec-1 x 10(-3)). The catalytic constant of the Y181I mutant was 6-fold higher and that of the Y188L mutant was 6-fold lower. Whereas TIBO displayed a linear mixed-type (noncompetitive) inhibition with respect to the deoxynucleotide substrate when wild-type p66/p51 was used, the pattern of inhibition became competitive or uncompetitive with Y181C or Y181I, respectively. Thus, the TIBO binding site of HIV-1 RT seems to be functionally and/or spatially related to the natural deoxynucleoside triphosphate binding site.
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PMID:Kinetics of different human immunodeficiency virus type 1 reverse transcriptases resistant to human immunodeficiency virus type 1-specific reverse transcriptase inhibitors. 768 49

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

Using 3D searching techniques based on algorithms derived from graph theory, we have established two previously unreported structural similarities involving the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT). First, we report that there is a strong similarity between the 3D folds of the RNase H domain of RT and the 'ATPase folds' of hexokinase, the 70 kDa heat-shock cognate protein and actin. Like RNase H, these enzymes are involved in nucleotide binding and metal ion-catalysed cleavage of a phosphodiester bond. Similarities of the folding motif and the position of the metal-binding site in these enzymes suggest possible functional analogies and evolutionary relationships with RNase H. Second, we find there is a strong resemblance between the folds of the RNase H domain and of the p66 and p51 'connection' domains of RT. It is possible that this striking similarity within the RT structure indicates a possible ancestral gene doubling event. The similarity may also indicate that the connection domains possess functional roles in addition to those previously suggested, and they may therefore represent a further target for the design of therapeutic agents.
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PMID:Three-dimensional structural resemblance between the ribonuclease H and connection domains of HIV reverse transcriptase and the ATPase fold revealed using graph theoretical techniques. 768 87

Packaging of the genomic RNA dimer and replication primer tRNA(Lys,3) into HIV virions are required for the production of infectious virus. The initiation of reverse transcription necessitates the annealing of tRNA(Lys,3) to the primer binding site (PBS) of HIV RNA by nucleocapsid (NC) protein. In this report the interactions of replication primer tRNA(Lys,3) with various forms of reverse transcriptase (RT) and nucleocapsid protein have been analyzed by ultraviolet light (UV) cross-linking and gel retardation assays. We show that of the three forms of RT studied, p66/p51, p66 and p51, only the heterodimer p66/p51 can tightly and stably interact with tRNA(Lys,3). Tight interactions between tRNA(Lys,3) and nucleocapsid protein, either NCp15 or NCp7, were found to take place within the anticodon domain. Interestingly enough, primer tRNA(Lys,3) can interact with RTp66/p51 and NCp15 to form a high molecular weight complex in which RTp66/p51 appears to enhance the binding of NCp15 to tRNA(Lys,3). These findings favor the notion that the RT enzyme and NC protein co-operate to select and package primer tRNA.
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PMID:Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. 768 91

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

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.
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PMID:Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli. 768 63

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

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4


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