Reverse transcriptase preferences

DNA polymerase, or reverse transcriptase—can be integrated into chromosomes of the mammalian cell. The integration of the animal vims DNA into the animal genome generally is not site-specific but does display site preferences. 

Adults The recommended dosage is 600 mg once daily in combination with a protease inhibitor or nucleoside analog reverse transcriptase inhibitors (NRTIs). It is recommended that efavirenz be taken on an empty stomach, preferably at bedtime. The increased efavirenz concentration following administration of efavirenz with food may lead to an increase in adverse events. 

Contemporary treatment of HIV infection requires the use of two nucleoside reverse transcriptase inhibitors (NRTI) combined with either a non-nucleoside reverse transcriptase inhibitors or a protease inhibitor. Which of the following NRTI backbones will you prefer to use in a patient recently diagnosed with HIV-associated dementia ... 

A sensitive method to measure toxic effects of drugs is to monitor cellular metabolism by incorporation of 3H-thymidine or 14C-protein hydrolysate. 14C-protein is used in preference to 3H-thymidine when it is necessary to avoid competition in uptake between the labeled thymidine and an unlabeled nucleoside reverse-transcriptase inhibitor. These methods are used mainly to monitor sublethal toxicity after initial screening or when comparing structure-activity relationships. The assays described here are carried out using 6-mL culture tubes (Note 1). These assays are carried out in parallel with the antiviral assay. 

Reverse transcription of RNA is sometimes used, e.g., for clone selection by colony hybridization (Verbeek and Tijssen, 1988). Although both avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptase can be used, we prefer the AMV enzyme if there are no strong secondary structures in the RNA (MMLV enzyme can be used at higher temperatures, even up to 55°C). The optimal pH range for the AMV enzyme is very narrow (should be 8.3) whereas the MMLV enzyme is active in a range of 7.6-8.3. Superscript reverse transcriptase from BRL is a cloned MMLV enzyme from which the RNase H activity has been deleted. 

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

AZT is particularly effective because the HIV reverse transcriptase actually prefers it over the normal nucleotide, thymidine. Nonetheless, AZT is not a cure. At best it prolongs the life... 

Efavirenz is used widely in the developed world because of its convenience, effectiveness, and long-term tolerability. To date, no antiretroviral regimen has produced better longterm treatment responses than any efavirenz-containing regimen in randomized, prospective clinical trials. As a result, efavirenz plus two nucleoside reverse-transcriptase inhibitors was one of two regimens preferred in 2004 for treatment-naive patients. 

In addition to NRTIs, the two other important classes of antiretroviral drugs available are the protease inhibitors and the nonnucleoside reverse transcriptase inhibitors (NNRTIs). Most authorities recommend initiation of antiretroviral therapy in treatment-naive patients with viral RNA loads above 10,(XX) copies/mL even if the CD4 cell count is normal. Preferred highly active antiretroviral therapy (HAART) combines one drug from the class of protease inhibitors with two NRTIs. Alternatively, one NNRTI may be combined with two NRTIs. 

Proteases have proven to be a favored target class for SBDD. Good crystal structures can often be obtained, substrate preferences are known, and the diseases involved (hypertension, HIV, HCV, etc.) are important ones with unmet needs. HIV protease was the second target to come along in AIDS after reverse transcriptase. Comparison of the HIV-1 viral genome with those of other retroviruses suggested that it too encoded for a protease. This protein, which proved to be necessary for the production of mature, infectious viral particles, was discovered to be an aspartyl protease smaller than those found in mammals, and it was proposed that it acted as a homodimer, which several X-ray structures of the enzyme published in 1989 confirmed. ... 

The primer-template preference of Pol D, as compared with other DNA polymerases including Pol BI, Taq polymerse, and Bacillus caldotenax (Bca) polymerase, is shown in Table II. Pol D prefers primer-extension type substrates to gap-filling type template-primers, such as the DNase I-activated DNA. This tendency is similar to the results from Taq polymerase and Bca polymerase, but in contrast to that of Pfu Pol BI. Pol D uses RNA primer more efficiently than other enzymes. Very little reverse transcriptase activity, comparable to the level of Taq polymerase, is detected. Figure 4 shows the excellent primer extension ability of Pol D. Here the in vitro primer extension rate, using the linearized M13 single-stranded DNA as a template, is about 4-5 bases/sec and 60 bases/sec for Pol BI and Pol D, respectively. 

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