Reverse transcriptase assay for analysis of resistance to anti-HIV drugs and their mechanism of action

Abstract

Reverse transcriptase (RT) is a viral enzyme and one of the main targets for drugs against human immunodeficiency virus (HIV). This work presents a set of sensitive non-radioactive RT assays were developed: standard RT inhibition assay (IC assay); RT binding inhibition assay; RTprotein detection assay and template-primer destruction assay or chain termination assay (CT assay). All are based on immobilized polyriboadenylic acid [poly(rA)] template in a 96-well microtitre plate, and use oligodeoxythymidylic acid (odT) as primer and 5- bromodeoxyuridine 5-triphosphate (BrdUTP) as substrate. The incorporated BrdU binds alkaline phosphatase conjugated anti-BrdU antibody that can be quantified by colorimetry or fluorimetry.

A set of substances with known mechanism of action were examined in the RT assays. Different types of inhibitors exhibited different behaviors in relation to their mode of action. The combined use of the assays was able to define which specific step of reverse transcription the inhibitor affected. The IC50 values obtained were similar to those reported for soluble RT assays, which indicated that the assays can be used for screening of RT inhibitory substances. The study of four non-nucleoside RT inhibitors (NNRTIs) 9-Cl-TIBO, Nevirapine, MSA-300 and Delavirdine showed that none of them prevented RT binding to template-primer. MSA-300 had a higher affinity for RT than the other NNRTIs tested.

The biochemical mechanism of resistance to AZT remained a puzzle for a long period. The CT assay and a modified IC assay that were supplemented with physiological concentration of GTP were applied for characterization of the RTs from 18 HIV-1 isolates with various susceptibilities to AZT in cell culture. Up to 9- fold and 600-fold variations in susceptibility to AZT-TP were revealed in IC assay and CT assay, respectively. The CT50 values had a tendency to increase with the occurrence of increased numbers of mutations associated with AZT resistance. The substitution T39A was observed in two isolates that were highly resistant to primer termination.

An RT purification procedure was developed to quantify and characterize RT from plasma. The procedure consists of three steps: pre-treatment of plasma to inactivate cellular enzymes; immobilizing virions on a gel and removing antiviral drugs, RT blocking antibodies by a wash; lysis of the immobilized virions and elution of viral RT. A study of 391 samples from HIV infected individuals showed a strong correlation between RT load and RNA copies obtained by PCR. The RT load assay could detect a broad range of different HIV subtypes.

Two phenotypic drug (NNRTIs and thymidine analogue chug) susceptibility tests were developed and used for characterization of RT derived directly from plasma. A high degree of concordance was found between the drug susceptibility profiles of plasma RTs and the occurrence of mutations associated with drug resistance. The assays are technically simple, rapid, and do not require complex interpretation of the results.

In conclusion, the presented non-radioactive RT assays proved useful for screening RT inhibitory substances, for dissection of the mode of action of RT inhibitors, and for characterization of drug susceptibility of RTs from various sources.