A NONRADIOACTIVE MICROTITRE PLATE REVERSE-TRANSCRIPTASE (RT) ASSAY, BASED ON IMMOBILIZED TEMPLATE, FOR SCREENING OF RT ACTIVITY INHIBITORS AND EVALUATION OF THEIR MODE OF ACTION

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjuga ted polyadenylic acid (prA), was investigated for simple analyses of t he RT inhibiting capacity and mode of action of various substances. Th ree different technical procedures using the assay were evaluated: (i) direct IC50 determinations with various substances, using four differ ent combinations of primer and dNTP amounts; (ii) analyses of the capa city of the substances to interfere with the binding of RT to template or template-primer (BIC50); (iii) analyses of the capacity of the sub stances to destroy the template-primer in presence or absence of RT (T DC50). The assay was found to be useful for all three purposes using s mall amounts of recombinant RT. In the IC50 analyses, the test substan ces gave values similar to those reported for soluble RT assays, and t he values varied in accordance with their known mode of action in rela tion to the combination of primer and dNTP amount used. Only one of th e substances, prG, in addition to DNA and RNA gave true RT binding inh ibition. The template destruction assay showed that chain terminating substances gave destruction at low inhibitor concentrations. Furthermo re, this destruction was RT-dependent, in contrast to the destruction obtained with substances that can base-pair with the template or prime r. For optimum information on mode of action of a given substance all three assay procedures should be used. The use of the assay in relatio n to the screening and analyses of new RT inhibitory substances and ch aracterization of RT in primary isolates or plasma is discussed.