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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.
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PMID:Morphogenesis of recombinant HIV-2 gag core particles. 152 43

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein. 154 Apr 16

We have cloned the simian foamy virus type 1 genome (SFV1) and determined its nucleotide sequence. Analysis of this genome reveals, in addition to the usual genes encoding retroviral capsid, reverse transcriptase, and envelope protein (respectively, gag, pol, and env), two open reading frames (ORFs) between env and the long terminal repeat with partial homology to the human foamy virus (HFV) bel1 and bel2 genes. The first ORF could code for a polypeptide of 312 amino acids (aa) showing 40% homology with the HFV bel1 putative gene product. A more detailed analysis showed that the protein encoded by this ORF would have features characteristic of known trans-activating proteins. The second ORF could code for a polypeptide of 403 aa showing 38% homology with the putative HFV bel2 gene product. Moreover, the 5' extremity of the RNA genome can be folded into a secondary structure identical to the Tat-response element of human immunodeficiency viruses. A phylogenetic tree of retroviruses, including SFV1 and HFV, was constructed. It showed at the molecular level that Spumavirinae, previously classified on the basis of their morphology and their biological properties, constitute a separate group. The homology between SFV1 and HFV reaches 89% in the reverse transcriptase domain of the pol gene. but is much smaller in other parts of the genome.
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PMID:Sequence analysis of the simian foamy virus type 1 genome. 164 58

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

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

Using mutational analysis, we have investigated the translation strategy of the reverse transcriptase gene (pol) of human hepatitis B virus. It has been proposed that this pol gene product is synthesized as a core-pol fusion protein from a polycistronic mRNA template via ribosomal frameshifting, a mechanism often seen in retroelements. Our data indicate that creation of a novel ATG initiation codon near the original ATG can compensate for a lethal missense mutation at the first ATG position of the pol open reading frame. Genetic analysis has rigorously ruled out the possibilities of frameshifting, non-ATG initiation, or RNA editing. These results are discussed in the context of a 5'-end entry model versus a novel model of direct internal entry of ribosomes.
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PMID:cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG. 168 89

We constructed a recombinant vaccinia virus carrying the entire gag and pol genes of human immunodeficiency virus type 1 (HIV-1). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of 100-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pol proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pol gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production.
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PMID:Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV. 168 17

T-cell-mediated cytotoxicity may play an important role in control of infection by the human immunodeficiency virus (HIV). In this study, we have identified and characterized a relatively conserved epitope in the HIV-1 reverse transcriptase recognized by murine and human cytotoxic T cells. This epitope was identified using a murine antigen-specific CD8+ class I major histocompatibility complex-restricted cytotoxic T-cell (CTL) line, a transfected fibroblast cell line expressing the HIV-1 pol gene, recombinant vaccinia viruses containing different truncated versions of the pol gene, and overlapping synthetic peptides. The optimal antigenic site was identified as residues 203-219 by synthesizing extended or truncated peptide analogs of the antigenic fragment. The optimal peptide was then tested for sensitization of autologous Epstein-Barr virus-transformed B-cell targets for killing by fresh human peripheral blood mononuclear cells. It was recognized by CTLs from several HIV-seropositive patients but not from any seronegative donor. Therefore, this peptide is a good candidate for inclusion in an AIDS vaccine. This study demonstrates that the same CTL epitope can be seen by murine and human CD8+ CTLs, as previously demonstrated for epitopes recognized by CD4+ helper T cells, and suggests the utility of screening for immunodominant CTL epitopes in mice prior to carrying out studies in humans.
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PMID:An epitope in human immunodeficiency virus 1 reverse transcriptase recognized by both mouse and human cytotoxic T lymphocytes. 169 Apr 29

Approximately 50% of the ribosomal DNA (rDNA) units of Drosophila melanogaster are inactivated by two different 28 S RNA ribosomal gene insertions (type I and type II). We present here the nucleotide sequence of complete type I and type II elements. Conceptual translation of these sequences revealed open reading frames (ORFs) encoding amino acid residues conserved in all retrotransposable elements. Full-length type I elements are 5.35 x 10(3) base-pairs in length and contain two overlapping ORFs. The smaller ORF (471 amino acid residues) has similarity to gag genes, while the larger ORF (1021 residues) has similarity to pol genes. Full-length type II elements are 3.6 x 10(3) base-pairs and contain one large ORF (1056 residues) that appears to represent a gag-pol fusion. Type I and type II elements are similar in structure, in the proteins they encode, and in insertion specificity to the R1Bm and R2Bm retrotransposable elements of Bombyx mori. We suggest that the D. melanogaster elements be called R1Dm and R2Dm, to reflect their structure as retrotransposons. Comparison of the R1 and R2 elements from these two widely different species revealed regions of the ORF that are likely to play an important role in the propagation of the elements. Four distinct regions of sequence conservation separated by regions of little or no sequence similarity were detected for both the R1 and R2 elements: (1) cysteine motifs of the gag gene, with three such motifs for R1 and one motif for R2; (2) a reverse transcriptase domain; (3) an integrase domain located carboxyl terminal to the reverse transcriptase region; and (4) a small region amino terminal to the reverse transcriptase domain, whose function is not known. The level of identity of the amino acid residues for these segments is 28 to 34% between the R1 elements, and 34 to 39% for the R2 elements. Finally, it may be predicted that the mechanism of unequal crossover might eventually eliminate R1 and R2 from the rDNA locus. The long history of selection at the protein level exhibited by these elements indicates that it is their active transposition that maintains them in the locus. The high level of sequence homogeneity between copies of each element within the same species is consistent with the high turnover rate expected to result from these processes.
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PMID:Type I (R1) and type II (R2) ribosomal DNA insertions of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori. 169 Aug 12

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.
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PMID:Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 Dalton subunit of reverse transcriptase. 169 40


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