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 virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.
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PMID:Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. 198 79

Cytoplasmic extracts prepared from cells infected with metabolically radiolabeled virions of human immunodeficiency virus type 1 contain viral DNA in association with labeled viral proteins. Viral DNA can be purified from these extracts by gel filtration chromatography and sucrose gradient sedimentation as a part of a nucleoprotein complex containing integrase as the only viral protein detectable by immunoprecipitation and gel electrophoretic analysis. The purified complex contains no detectable gag gene products, including p17, p24, p7, or p6, and contains no additional pol gene products, including the p10 protease, p66 and p51 polymerase, or the p15 RNase H. Nearly all of the purified nucleoprotein complexes are capable of integrating into heterologous DNA targets in vitro. These observations demonstrate that integrase is a component of the human immunodeficiency virus type 1 preintegration complex and suggest that integrase may be the only viral protein necessary for the integration of retroviral DNA.
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PMID:Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. 200 49

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

Human endogenous retroviral element S71 had previously been shown to contain gag- and pol-related regions and a 3' LTR-like sequence. The nucleotide sequence of S71 was determined and compared with the corresponding regions of SSV and its helper virus SSAV. The 1.48-kb S71 gag region consists of matrix protein p15 (MA)-, capsid protein p30 (CA)-, and nucleocapsid protein p10 (NC)-related sections and the 1.82-kb pol region of tether, RNase H (RH), and endonuclease/integrase (IN) sections. The S71 nucleotide sequence contains a 167 amino acid open reading frame encompassing MA. The boundaries of the S71 element are delimited by direct repeats and the entire element is 5.4 kb long. Similarity between S71 and the v-sis-bearing, defective SSV provirus also covers overall structural organization, including the presence of presumably nonretroviral sequences. Both the gag and the pol regions of S71 contain sequences highly conserved in numerous retroviruses. Phylogenetic analysis with conserved CA, RH, and IN sequences showed that of all other (C-type) human retroviral elements available for comparison, S71 is most closely related to infectious primate and murine retroviruses. This suggests that S71 represents a phylogenetic subgroup of its own. In addition we identified short ranges of conserved amino acid sequences within C-type retroviral gag and pol genes sufficient for phylogenetic analysis. Use of these may facilitate large-scale phylogenetic evaluation of C-type retroviral elements and allow rapid classification of new elements.
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PMID:S71 is a phylogenetically distinct human endogenous retroviral element with structural and sequence homology to simian sarcoma virus (SSV). 215 93

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
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PMID:Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. 245 55

Plant ribosome-inhibiting proteins are shown to be homologous at the domain level to RNase H from Escherichia coli and to two regions of the pol gene product of retroviral reverse transcriptases. One of these regions carries the viral integrase or int function, while the other has previously been suggested to contain the viral RNase H exo activity. Several residues conserved among the ribosome inhibitors, E. coli RNase H, and the integrase proteins are seen to occupy a prominent cleft in the tertiary structure of the ribosome inhibitor ricin, suggesting roles in binding or catalysis. It is likely that these homologous sequences represent modern derivatives of an ancient protein-folding unit capable of nucleic acid binding and modification which has been incorporated into a variety of enzyme functions.
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PMID:Ribosome-inhibiting proteins, retroviral reverse transcriptases, and RNase H share common structural elements. 245 78

The lily retrotransposon del 1-46 is 9345 base pairs (bp) long. It has long terminal repeats (LTRs) of 2406 bp (left) and 2415 bp (right), which differ in sequence by 1.4%. Sequences similar to those involved in priming DNA synthesis in retroviruses occur in the internal region. Near the left LTR is a sequence complementary to 18 residues at the 3' end of methionine initiator tRNA of three plant species, and a run of 12 purines occurs close to the right LTR. One internal reading frame of del 1-46 has relatively few stop codons. The 1462-codon product from this frame has motifs, in N to C terminus order, corresponding to those identified with RNA binding, protease, reverse transcriptase, RNase H, and integrase functions in retroviruses and certain other retrotransposons. Amino acid sequence comparisons of three conserved pol regions show del to be closely related to the Ty3 retrotransposon of yeast (37-40% identity). del is also related to the gypsy group of Drosophila (17.6, 297, gypsy/mdg4, and 412), showing closer identity with their reverse transcriptase (32-38%) and RNase H (36-45%) domains than with their integrase domain (21-26%). It is proposed that a gypsy group ancestor exchanged the integrase region with a more distantly related element since its divergence from a del/Ty3 common ancestor. The occurrence of related retrotransposons in three different kingdoms (plants, animals, and fungi) strongly implies their horizontal transmission in recent evolutionary time.
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PMID:Plant retrotransposon from Lilium henryi is related to Ty3 of yeast and the gypsy group of Drosophila. 254 87

Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner. IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA. Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif. We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 A resolution. The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed beta-sheet region surrounded by five alpha-helices. Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H. The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 A resolution. However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered. The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV.
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PMID:High-resolution structure of the catalytic domain of avian sarcoma virus integrase. 756 93

The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.
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PMID:Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. 762 12

Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.
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PMID:The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides. 768 36


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