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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.
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PMID:Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. 171 88

Replication of human immunodeficiency virus 1 (HIV-1) uses a viral reverse transcriptase (RT) to convert its positive-strand RNA into double stranded DNA, which is then integrated into host genome. Reverse transcription is a complex event involving p66 and p51 RT subunits but also several viral proteins including Nef, Tat, Vif, IN, NCp7 and p55gag. Viral RNA itself forms a primer/template complex by association with a cellular tRNA(Lys3) which is already present in mature virions. A RT initiation complex (RTIC) is thus formed which may also involve cellular protein upon viral entry. X rays diffraction and NMR studies of free or inhibitor-bound RT have led to the recognition of RT 3D structure, and allowed a thorough understanding of the mode of action of classical competitive nucleoside RT inhibitors (NRTIs) and of the binding of allosteric, non NRTIs (NNRTIs) inhibitors. This also opened an access to computer-aided drug design and modeling. Current NNRTIs represent, in terms of chemical structures, a heterogeneous class of inhibitors currently undergoing extensive development. By contrast with NRTIs, they seem to block initiation steps of reverse transcription. Molecular dynamics, detailed analysis of their interaction with RT as well as the incidence, in the series, of cases of non classical biological behavior, as illustrated here for a new family of compounds, suggest mechanisms of action which are not understandable without considering the involvement of the RTIC as a whole. This opens the exciting perspective of developing new compounds based on this integrated knowledge. Key Words: Nonnucleoside reverse transcriptase inhibitors (NNRTIs); Reverse transcriptase initiation complex (RTIC); Human immunodeficiency virus (HIV); Non classical nonnucleoside reverse transcriptase inhibitors; Molecular modeling; Docking; QSAR; Natural endogenous reverse transcription (NERT).
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PMID:Nonnucleoside inhibitors of HIV-1 reverse transcriptase: from the biology of reverse transcription to molecular design. 1452 23

Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.
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PMID:Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. 1511 87

The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in (1171)Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH(2)-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
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PMID:Proteolytic processing of sapovirus ORF1 polyprotein. 1591 82

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.
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PMID:Cross-clade inhibition of recombinant human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVcpz reverse transcriptases by RNA pseudoknot aptamers. 1732 28

Protein-protein interactions are crucial to biological functions. Consequently, designing drugs to control protein-protein interactions is receiving increasing attention. Protein structures can associate in different ways. Analysis of the structures of protein-protein complexes using amino acid sequence order-independent multiple structural comparison algorithms, led us to conclude that the amino acids Trp, Met, and Phe are important for protein-protein interactions. Hence, in principle, drug design targeting the Trp/Met/Phe should modulate protein functions effectively. Several clusters of the Trp/Met/Phe residues are involved in the p53 protein-protein interactions. The best example in this regard is the Phe19/Trp23 of p53, which binds to transcriptional factors and to the MDM2 protein. In the HIV related proteins, the Trp/Met/Phe residues have roles in the dimerization of the transcriptase (p51/p66) and in cell-fusion processes, including the gp120-CD4 interaction and the gp41 six-helix bundle formation. Trp/Met/Phe residues are preferred in 'normal' functional protein-protein interactions and they also appear to be exploited in amyloid formation, especially the phenylalanine. Comparison of binding propensity and amyloid formation preference reveals that apart from Lysine, Isoleucine is the least structurally conserved in protein binding sites and has a high propensity in sequences forming amyloids. Thus, this may suggest that nature tends to avoid Ile conservation in protein-protein interaction to avoid amyloid formation. In this regards, Trp/Met/Phe as well as Ile may be targeted to modulate protein-protein interaction.
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PMID:Trp/Met/Phe hot spots in protein-protein interactions: potential targets in drug design. 1750 33

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