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 gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.
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PMID:Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. 168 46

As human immunodeficiency virus type 1 (HIV-1) has become better understood, numerous drugs have been developed that act at virus-specific sites. These are challenging our ability to evaluate them thoroughly and rapidly. Zidovudine (AZT) remains the mainstay of anti-HIV-1 drugs. Recent controlled trials indicate it should be used early in infection (in those with CD4 cell counts less than 500/mm3) and in lower doses (500-600 mg/day). Prolonged AZT treatment in patients with AIDS, however, is often associated with viral resistance. Newer reverse transcriptase-inhibiting nucleoside derivatives are currently in phase II-III clinical trials. Other HIV-1 replicative sites under attack in clinical studies include binding and entry of virus, envelope protein glycosylation, and viral assembly and release. Agents that target HIV-1 proteinase, integrase, ribonuclease H, and products of regulatory genes such as tat are under development. Combination therapies that target different viral replicative sites likely will allow use of individual agents below their toxic concentrations and help prevent drug resistance. Innovative programs for expanded access to experimental drugs are needed that will permit expeditious clinical trials, optimize the gathering of useful information, and permit the widest access to promising treatments.
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PMID:Chemotherapy of human immunodeficiency virus infections: current practice and future prospects. 169 Dec 43

We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay. Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule. With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function. This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT. Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus. We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties. Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.
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PMID:Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. 169 64

We have analyzed the processing of the RNA primer for (+) strand DNA synthesis by reverse transcriptase of the human immunodeficiency virus 1. To test for specific RNA cleavage and primer usage, we constructed a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking viral sequences. Although the RNase H activity of reverse transcriptase cleaves the RNA strand into multiple fragments, only two primers are extended in the presence of nucleoside triphosphates. The major RNA primer includes the entire polypurine tract except for the last adenosine and has the sequence 5'-UUUUAAAAGAAAAGGGGGG-3'. The minor primer has the same 3' end but is two nucleotides shorter. In a subsequent processing step reverse transcriptase releases the primer intact via a cleavage at the RNA-DNA junction. RNA cleavage, primer extension, and primer removal can take place in a single reaction. However, specificity does not require coupling of the three steps and is preserved in the individual reactions. The polypurine primer is generated and removed after its elongation in the absence of DNA synthesis. Furthermore, the polypurine primer is selected among the several RNA fragments available and extended by reverse transcriptase as well as by p51, a short form of reverse transcriptase lacking RNase H activity.
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PMID:Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. 169 20

We have analyzed the effects of several natural compounds related to avarols and avarones on the catalytic functions of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The most potent substances, designated as avarone A,B and E and avarol F, inhibited indiscriminately the enzymatic activities of HIV-1 RT, namely the RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H. The inhibition of the DNA polymerase activity was found to be non-competitive with respect to either the template-primer or the deoxynucleotidetriphosphate. These studies suggest that the hydroxyl group at the ortho position to the carbonyl group at the quinone ring is involved in blocking the RT activity. The identification of the active site of the inhibitors will hopefully lead to the rational design of new potent anti-HIV drugs.
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PMID:The inhibition of human immunodeficiency virus type 1 reverse transcriptase by avarol and avarone derivatives. 169 11

The sequence of the LTR-LTR circle junction of human immunodeficiency virus type 1 (HIV-1) was determined. The circle junction sequences were amplified by the polymerase chain reaction and cloned into M13 sequencing vectors. The circle junction contains 4 base pairs that are not present in the integrated provirus. We show that reverse transcription in HIV-1 initiates with the addition of a dC to the tRNA primer, suggesting that the tRNA used to initiate reverse transcription ends with the consensus CCA triplet. This indicates that the source of one of the four bases in the circle junction is probably the terminal A of the tRNA primer used to initiate reverse transcription. We propose that, in HIV-1, removal of the tRNA primer by RNase H cleavage shows an unusual specificity such that cleavage occurs between the terminal rA and the adjacent rC of the tRNA primer. These data also imply that the HIV-1 integration protein removes two bases from each end of the linear viral DNA during integration as has been described for other well-studied retroviruses.
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PMID:Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration. 169 9

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.
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PMID:Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization. 169 57

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.
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PMID:Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast. 169 61

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.
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PMID:HIV-1 reverse transcriptase: structure predictions for the polymerase domain. 170 98

Human immunodeficiency virus reverse transcriptase.RNase H (RT.RNase H) has an RNA hydrolysis specificity which was influenced both by the sequence of the DNA primer-RNA template and by binding of the polymerase active site to the primer 3' terminus. RT.RNase H selectively hydrolyzed the phosphodiester bond between the 15th and 16th ribonucleotide back from the ribonucleotide that is complementary to the primer 3'-terminal deoxynucleotide. The cleavage site for RT.RNase H remained a fixed distance behind the 3'-primer terminus as the polymerase extended the primer. This cleavage was not strongly affected by the position of the DNA primer on the template nor was it affected by reducing the primer length from 40 to 25 nucleotides. These results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 15-16-nucleotide DNA-RNA heteroduplex. Since one helical turn is approximately 10 nucleotides, the distance between the active sites is 1.5 helical turns of heteroduplex. Therefore, the two active sites (catalyzing reactions on opposite strands) bind the same side of the RNA-DNA double helix. RT.RNase H also showed some sequence dependence for the site of hydrolysis. This sequence dependence has not been fully characterized. The rate of RT.RNase H cleavage was weakly inhibited by the next coded deoxynucleoside triphosphate following the incorporation of a dideoxynucleotide.
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PMID:Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. 170 25


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