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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)

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.
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PMID:On the early emergence of reverse transcription: theoretical basis and experimental evidence. 128 61

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
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PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

To establish an animal model of AIDS, two different "wild" or "adapted" HIV2 Rod and Eho strains were cultivated on monkey cells from different species (baboons, cynomolgus, Rhesus monkeys). Five different available strains were then injected both by intravenous (i.v.) and intracerebral (i.c.) route into ten Rhesus monkeys. Seven animals seroconverted between days 13 and 230. Reverse transcriptase activity in the lymphocyte culture supernatants was detectable in six of the seven animals that seroconverted, and in one animal that remained seronegative. Lymphopenia and a decrease in the CD4+ cell counts were observed in eight animals. One animal, inoculated with HIV2-Rod "wild type," developed a severe cachexia, with dyspnea, and associated neurological symptoms 150 days after inoculation. This animal was sacrificed on day 220. Pathological examination showed typical lesions of actinomycetes infection in the lungs and in the meninges. Another monkey had significant weight loss associated with lymphadenopathies and pancytopenia. These results suggest that in vivo replication of HIV2 in Rhesus monkeys may induce clinical symptoms of immune deficiency. This method is reproducible and may provide a good model for AIDS.
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PMID:Clinical and virological aspects of HIV2 infection in rhesus monkeys. 147 23

A culture of rhesus monkey peripheral blood lymphocytes was divided into two parts; one was kept as an uninfected control, and the other was infected with a strain of simian immunodeficiency virus (SIVmac251) originally isolated from a rhesus monkey that died of a malignant lymphoma associated with acquired immune deficiency syndrome. Both cultures were sampled at successive intervals from 1 to 40 days postinfection. Each sample was subjected to in situ hybridization for detection of viral mRNA, immunocytochemical detection of viral core protein (p27), reverse transcriptase assay, electron microscopy, and immunophenotypic characterization of infected cells. These techniques were used to define viral growth kinetics of this novel lentivirus in peripheral blood lymphocytes. The first evidence of SIVmac251 replication was obtained by an in situ hybridization signal for viral mRNA at 2 days postinoculation. This was followed by detection of viral p27 core protein by immunocytochemistry on day 4. Reverse transcriptase activity above control values was not detected until day 8. Budding particles were not found in the infected cultures until 14 days postinfection. Results of in situ hybridization, immunocytochemistry, and reverse transcriptase assay indicated that two bursts of viral replication occurred during the course of this study. The first, at 3 weeks postinfection, was due to infection and subsequent depletion of CD4+ lymphocytes, while the second, 3 weeks later, resulted from a cycle of replication in CD8+ lymphocytes and the remaining CD4+ cells, culminating in the death of all cells on day 39 postinoculation.
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PMID:Study of long-term cultures of simian immunodeficiency virus (SIVmac 251)-infected peripheral blood lymphocytes. 169 33

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.
AIDS Res Hum Retroviruses 1990 Sep
PMID:HIV-1 reverse transcriptase: structure predictions for the polymerase domain. 170 98

Reverse transcriptase (RT) transcribes viral RNA into DNA to be integrated into the host genome. To study epidemiological aspects of human leukemias and lymphomas which are known to express retroviruses, clinical specimens in this report were assayed for divalent cation-dependent viral-specific RT. The assay was carried out with cells solubilized with a detergent to release RT enzyme. RT was purified with poly(U)-Sepharose which fixed all DNA polymerases and assayed with 4 synthetic homopolymers, oligonucleotide primed-templates, poly(rA)-oligo(dT)12-18 or poly(dA)-oligo(dT)12-18 with Mg2+, poly(rC)-oligo(dG)12-18 or poly(rCm)-oligo(dG)12-18 with Mn2+ as divalent cation and [methyl-3H]thymidine 5'-triphosphate or deoxy[8-3H]guanosine 5-triphosphate respectively. Radioactivity incorporation of the precipitate allows quantitation of RT activity. One Hodgkin's disease, one out of 2 B lymphomas, one out of 2 T lymphomas, eight out of 12 leukemias were found to be positive for RT activity as well as acquired immunodeficiency syndrome (AIDS) patients, known to express RT. The obtained RT activity in hematological malignancies was found to be comparable to positive controls such as RT enzymes purified from avian myeloblastosis and Moloney murine leukemia viruses.
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PMID:Presence of reverse transcriptase in human leukemias and lymphomas. 170 70

Biological properties of an AIDS agent first isolated from a native citizen in the USSR are presented. The source of the virus was a young Byelorussian woman who in the near past had had sexual contacts with a citizen from one of the Central Africa countries. The isolate is thought to be of HIV-I type. It replicated perfectly in many continuous lymphocyte lines and had HIV-characteristic morphology. The protein spectrum of the isolate was gp120, gp41, p65/51, p55, p32, p24, p17. Reverse transcriptase activity was detected in the culture fluid of the virus-containing cell cultures. The isolate was designated HIV-IZ.
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PMID:[The biological properties of the HIV isolated from a virus carrier living in the Byelorussian SSR]. 214 58

Reverse transcriptase activity of the human immunodeficiency virus (HIV) was blocked in vitro by immunoglobulin G (IgG) derived from certain individuals infected with this retrovirus. A heterogeneous immune response for inhibition of enzyme function was noted. Catalytic activity was depressed by 50% or more with the use of 10 micrograms of IgG from 11 of 16 HIV-seropositive asymptomatic carriers, but from 0 of 8 seronegative controls and 2 of 12 patients with acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The inhibitor was confined to the F(ab')2 fragment. It was not directed against the poly(rA) X oligo(dT) template, nor against major envelope or structural viral antigens, and did not cross-react with bacterial, avian, or other mammalian DNA polymerases. It did not correlate with recognition of polymerase antigens by radioimmunoprecipitation. Loss of this inhibitor may be associated with development of clinical disease. Ten asymptomatic HIV-seropositive carriers with high titers of IgG antibodies to reverse transcriptase were followed for a mean of 3 years. All of four lost inhibitory capability prior to development of AIDS or ARC, while titers persist in the six who remain clinically healthy.
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PMID:Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. 243 4

In vitro experiments were conducted to assess whether bedbugs (Cimex lectularius and Cimex hemipterus) and mosquitoes (Aedes aegypti formosus) could act as vectors of HIV. These insects engorged through a membrane on a blood-virus mixture. Female bedbugs were larger than males and took larger blood-meals when fed to repletion. It was determined that the full blood-meal of a female bedbug contained 0.09 x 10(5) tissue culture infectious doses (TCID) of virus and a male 0.07 x 10(5) TCID, while partial meals taken when feeding was interrupted contained 0.013 x 10(5) TCID and 0.015 x 10(5) TCID for female and male bugs, respectively. Reverse transcriptase activity was assayed after culture of insect extracts in H9 cells: this showed survival of virus in C. lectularius for up to 4 h, in C. hemipterus for up to 1, possibly 2 h, but no survival in Ae. aegypti formosus. Four attempts to transmit the virus by interrupted feeding by C. lectularius from a blood-virus mixture to uninfected blood failed. It is concluded that Ae. aegypti formosus and probably other mosquitoes are not mechanical vectors of HIV and that such transmission is also unlikely to occur in bedbugs under natural conditions.
AIDS 1987 Sep
PMID:Experimental assessment of bedbugs (Cimex lectularius and Cimex hemipterus) and mosquitoes (Aedes aegypti formosus) as vectors of human immunodeficiency virus. 245 May 52

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.
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PMID:Fidelity of HIV-1 reverse transcriptase. 246 Sep 24


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