Reverse transcriptase activity assay

Figure 2. Elution profiles of both microvesicle-contaminated HIV-1lav-i preparation and microvesicle, protein profiles of subtilisin-treated HIV-1lav-i preparation and microvesicles, and HIV-1 reverse transcriptase activity assay.

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

The right start. Suppose that you want to assay reverse transcriptase activity. If polyriboadenylate is the template in the assay, what should you use as the primer Which radioactive nucleotide should you use to follow chain elongation ... 

Ultrasensitive Detection of Reverse Transcriptase Activity by the Amp-RT Assay... 

From the Fijian sponge Fascaplysinopis reticulata were isolated two sesterterpenoids related to luffariellolide, wodehydro-luffariellolide (69) and dehydro-luffariellolide diacid (70). Ao-dehydro-luffariellolide inhibited at 1 mg/ml 81% of the HIV-1 reverse transcriptase activity [90] and reduced the activity of p56lck tyrosine kinase at 0.5 mM to 45% in ELISA based assays [91]. Hyrtiolide (71) was isolated from the Fijian sponge Hyrtios erecta together with its correlated wo-dehydro-... 

Nucleotide Incorporation Assay Modification Measuring Reverse Transcriptase Activity... 

Krebs, J. F. Kore, A. R. Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues. Bioconjugate Chem. 2008, 19, 185-191. 

Identification of the Isolate. Growth of HIV isolates in cell culture is monitored once or twice weekly by measuring reverse transcriptase (RT) activity or the production of viral p 24 core protein by enzyme immunoassay of the culture medium. Most laboratories have established cutoff values for each assay and also monitor rises in RT activity or antigen level. 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

Cys202 [208]. Human immunodeficiency virus (HIV) is the primary cause of acquired immunodeficiency syndrome (AIDS). In an effort to find new drugs preventing the growth of HIV, Masao et al developed an in vitro assay method of RNase H activity associated with reverse transcriptase (RT) from HIV-1. Some 1,4-naphthoquinones moderately inhibited RNase H activity [209]. Several natural occurring naphtoquinones have showed antiretroviral activity [210-211],... 

Detection kit for HIV reverse transcriptase (RT) activity (e.g., Lenti-RT, Cavidi Tech., Upsala, Sweden) or p24 antigen (e.g., Coulter). Alternatively an in house P24 assay may be utilized (44). 

Adding compounds at increasing times postinfection (1.5-48 h) will also discriminate between compounds that act either early or late in the replication cycle of HIV. Reverse-transcriptase inhibitors will lose activity after about 6 h postinfection, whereas proteinase inhibitors will still retain significant activity at 48 h postinfection (references). The methodology is the same as that described in Subheadings 3.2. and 3.2.1. These experiments are typically only carried out with a single compound of interest together with an appropriate reference compound. A number of replicate assays are set up and dilutions of the compound added at 2, 4, 6, 12, 24, and 48 h. Dose responses are drawn for each time-point or responses are shown for each concentration at all time-points (7). 

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. 

Silver, J., Maudru, T., Fujita, K., and Repaske, R. (1993) An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions. Nucleic Acids Res. 21, 3593,3594. 

The tetrasodium salt of carbonylbisphosphonate was originally synthesized by Quimby et al. [76], using hydrolysis of a tetraalkyl dichloromethylenebisphos-phonate in aqueous NaOH. In aqueous solution, the yellow ketone form reversibly converts to its colorless hydrate at acidic pH [76], a process which can be assessed by a combination of 31P-NMR and uv-visible spectroscopy at pH 7, the ketone predominates [93]. The salt moderately inhibits HIV reverse transcriptase in a p24 assay, whereas the parent methylene compound is inactive [64], displays some activity vs the pyrophosphate-dependent phosphofructose kinase of the parasite T. gondii [94] and has found use as a selective inhibitor of PCNA-independent DNA polymerase 6, allowing its enzyme activity to be distinguished from that of DNA polymerase a [95]. 

Berberine chloride was evaluated in the human immunodeficiency virus reverse transcriptase assay and found to be moderately active (50 pg/ml < IC50 < 150 pg/ml)[229]. 

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