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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.
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PMID:Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity. 170 63

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.
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PMID:Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. 170 27

Screening of pharmacologically acceptable prototype compounds has recently led to the discovery of a series of ultraselective inhibitors of human immunodeficiency virus (HIV)-1 replication, the tetrahydroimidazo[4,5,1-jk] [1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives. The TIBO compounds completely suppress the formation of proviral DNA in acutely infected cells, as revealed by polymerase chain reaction (PCR) analysis. TIBO derivatives are inhibitory to the reverse transcriptase (RT) of HIV-1 but not that of HIV-2 or other retroviruses. The inhibition is most effective with poly(C)-oligo(dG) as the template/primer, and it is selectively directed against the RNA-dependent DNA polymerase activity and not the accompanying DNA-dependent DNA polymerase and ribonuclease H activity of HIV-1 RT. Kinetic studies point to an uncompetitive inhibition with regard to the template/primer. TIBO compounds are active against HIV-1 replication through a unique interaction with HIV-1 RT. The experimental data indicate the existence of a target on HIV-1 RT that is responsible for the inhibition of replication and a mode of action unrelated to that of previously studied RT inhibitors.
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PMID:An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and -thione derivatives. 170 38

We studied the effect of the natural marine substance illimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 microgram/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6'-hydroxyl group at the ortho position to one of carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity.
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PMID:Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. 170 12

The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural-functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied.
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PMID:Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 170 12

The complete pol region of the simian immunodeficiency virus from African green monkeys was cloned and expressed in E. coli. The reverse transcriptase was purified to high specific activity and could be shown to contain both reverse transcriptase activity as well as an associated RNase H activity. As is observed with other reverse transcriptases the enzyme is composed of two subunits which cannot be separated by conventional techniques. When comparing the recombinant enzyme with the authentic enzyme isolated from virus no differences were found by biochemical, enzymological, or immunological criteria. Moreover, the action of inhibitors against this enzyme did not show significant differences when compared to reverse transcriptases from HIV-1 and HIV-2.
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PMID:Cloning and expression of the complete SIVagm pol region in E. coli. Purification and partial characterization of the reverse transcriptase. 170 22

The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.
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PMID:Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. 170 86

Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed.
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PMID:Structural models of ribonuclease H domains in reverse transcriptases from retroviruses. 170 92

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.
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PMID:Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products. 171 Dec 3

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.
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PMID:The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity. 171 Dec 22


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