Reverse-transcriptase

This is the enzyme responsible for the synthesis of mitochondrial DNA and the DNA of some viruses, such as adenoviruses. Polymerase -y is very large and consists of a tetramer of identical oligomers, each having a molecular weight of 47,000. Synthetic ribonucleotides are very effective templates in the laboratory, but this mitochondrial enzyme differs from reverse transcriptase in that natural RNAs are poor templates. 

This enzyme is associated with the virions of RNA tumor viruses such as the Rous sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase. 

Eukaryotic chromosomes, unlike their bacterial counterparts, are linear rather than- circular. Since RNA oligonucleotides prime both prokaryotic and eukaryotic DNA synthesis, the 5 termini of the daughter 

Molecular weight 155,000, plus three other subunits 43,000 193,000 (four oligomers) 

Template specificity Nicked DNA template, RNA primer Nicked DNA template, DNA primer Ribonucleotide template and DNA primer 

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Eriksson M A L, J Pitera and P A Kollman 1999. Prediction of the Binding Free Energies of New TIBO-like HIV-1 Reverse Transcriptase Inhibitors Using a Combination of PROFEC, PB/SA, CMC/MD, and Free Energy Calculations. Journal of Medicinal Chemistry 42 868-881. 

The viruses responsible for AIDS are human immunodeficiency virus 1 and 2 (HIV 1 and HIV 2) Both are retroviruses, meaning that their genetic material is RNA rather than DNA HI Vs require a host cell to reproduce and the hosts m humans are the T4 lymphocytes which are the cells primarily responsible for inducing the immune system to respond when provoked The HIV penetrates the cell wall of a T4 lymphocyte and deposits both its RNA and an enzyme called reverse transcriptase inside There the reverse transcriptase catalyzes the formation of a DNA strand that is complementary to the viral RNA The transcribed DNA then serves as the template from which the host lymphocyte produces copies of the virus which then leave the host to infect other T4 cells In the course of HIV reproduction the ability of the T4 lymphocyte to reproduce Itself IS compromised As the number of T4 cells decrease so does the body s ability to combat infections... 

Reverse transcriptase inhibitors are also used against certain viruses which although they are not retroviruses do require re verse transcriptase to repro duce The virus that causes heptatitis B is an example... 

Section 28 13 HIV which causes AIDS is a retrovirus Its genetic matenal is RNA instead of DNA HIV contains an enzyme called reverse transcriptase that allows Its RNA to serve as a template for DNA synthesis m the host cell... 

Reverse transcriptase (Section 28 13) Enzyme that catalyzes the transcription of RNA to DNA Ribozyme (Section 28 11) A polynucleotide that has catalytic activity... 

The phosphorodithioates DNA derivatives have been shown to bind specifically to complementary DNA or ENA sequences to form stable adducts. Because they are also highly resistant to degradation by cellular exonucleases, these derivatives can be useful both for appHcations in research and as therapeutic dmgs. Phosphorodithioate DNA has been shown to stimulate Rnase H activity in nuclear cell extracts and is a potent inhibitor of HIV type-1 reverse transcriptase (56). 

The streptovaricins inhibit the reverse transcriptase of some RNA oncogenic vimses that may be involved in the process of viral transformation (see Antiviral agents). The atropisostreptovaricins again have similar activities to the corresponding natural isomers. The streptovals and streptovarone exhibit gready improved activity against reverse transcriptase relative to the streptovaricins (85), but their in vitro activities were low (86). The damavaricins also inhibit reverse transcriptase (4) as well as tumor cell growth (87). 

Rifampicin has also shown antiviral activity but at levels 500—1000 times greater than required for antibacterial activity (130,140—142). Rifampicin shows promise in the treatment of leprosy (130,143). A large number of rifampicinlike derivatives are potent inhibitors of reverse transcriptase (123,144-148). 

The deterrnination of the presence of reverse transcriptase in vims-infected cells can be done using labeled nucleotide triphosphates. Reverse transcriptase is an enzyme capable of synthesizing DNA from RNA and it is thought to play an important role in vims-mediated cell modification. This enzyme is also a marker enzyme for HIV, the vims impHcated in causing acquired immunodeficiency syndrome (AIDS). The procedure utilizes radiolabeled nucleotides with nonlabeled substrates to synthesize tagged DNA. The degree of radioactive incorporation reflects the reverse transcriptase activity. 

RNA-dependent DNA polymerase (reverse transcriptase) oncomavims, human immunodeficiency vims (HIV)... 

Basically, AZT is anabohcaHy phosphorylated to AZT mono-, di-, and tri-phosphates by various enzymes (kinases) of a target ceU (159). AZT-triphosphate competes with other phosphorylated pyrimidine nucleosides for incorporation into HIV DNA by the viral reverse transcriptase. Incorporation of the AZT-triphosphate into reverse transcriptase results in viral DNA chain termination. Reverse transcriptase is essential in the repHcative cycle of HIV. 

AH 2/3 -dideoxynucleoside analogues are assumed to be intraceUularly phosphorylated to thek active form (5 -triphosphate), and then targeted at the vims-associated reverse transcriptase. The rate and extent of the 2 /3 -dideoxynucleosides phosphorylate to the 5 -triphosphates may be of equal or greater importance than the differences in the relative abiUties of these 5 -triphosphates to inhibit the vkal reverse transcriptase (171). At the level of vkal reverse transcriptase, the 5 -triphosphate of AZT and other dideoxynucleosides may either serve as a competitive inhibitor with respect to the natural substrates or may act as an alternate substrate, thus leading to chain termination (172). 

The mode of action of PMEA may be quite similar to the mechanism by which (3)-HPMPA accomplishes its selective inhibitory activity against herpes vimses. Eor PMEA to reach its active triphosphate form, it needs only two phosphorylation steps. The triphosphate derivative of PMEA has a much stronger affinity for HIV-1 reverse transcriptase than for cellular DNA polymerases (175). Whether it is actually incorporated into DNA and terminates the growing DNA chain is currentiy under investigation. 

A recent example is the substrate analogue thymidine 5 -[a,P-iaiido]triphosphate [141171-20-2] (TMPNPP) (2) which competitively inhibits the human iaimunodeficiency vims-1 (HIV-1) reverse transcriptase (HIV-1 RT) with a iC value of 2.4 micromolar ]lM) (9). The substrate is thymidine 5 -triphosphate... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

RH Smith Jr, WL Jorgensen, J Tirado-Rives, ML Lamb, PAJ Janssen, CJ Michejda, MBK Smith. Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations m a linear response method. J Med Chem 41 5272-5286, 1998. 

Other nucleosides such as 2, 3 -dideoxyinosine (ddl) also block the action of reverse transcriptase and are often combined with AZT in drug cocktails. Using a mixture of drugs makes it more difficult for a virus to develop resistance than using a single drug. 

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Reverse transcriptase (Section 28.13) Enzyme that catalyzes the transcription of RNA to DNA. 

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