A novel, cell-free PCR-based assay for evaluating the inhibitory activity of antiretroviral compounds against HIV reverse transcriptase.
This study describes a novel, PCR-based assay that evaluates the ability of compounds to inhibit cDNA generation by HIV reverse transcriptase (RT), of both commercial and viral lysate origin, from a known RNA template. The template consisted of RNA from stable transfectants ectopically expressing the US6 gene of herpes simplex virus-1, coding for glycoprotein D. Controls were carried out to demonstrate that no residual DNA polymerase activity or DNA contamination was responsible for the amplified DNA in the tested, control samples. In this assay, 0.1 μM nevirapine totally inhibited the RT activity of 0.5 U commercial HIV RT, while 10 nM inhibited it by only 10%. Conversely, 10 pM efavirenz completely inhibited 0.5 U HIV RT. Similar results were obtained when a self-prepared viral lysate was used as a source of HIV RT. A reference commercial kit directly measuring HIV RT activity, without amplification, was less sensitive than the new assay. As a consequence, the HIV RT 50% inhibitory concentration of nevirapine and efavirenz in the newly described assay was 8 and 5 × 10(3) times lower, respectively, than in the commercial assay. In conclusion, this novel method was sensitive, reproducible, and sufficiently rapid for screening in vitro the functional activity of known or potential antiretroviral compounds against HIV RT.
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
Caterina
C
Frezza
Emanuela
E
Balestrieri
Francesca
F
Marino-Merlo
Antonio
A
Mastino
Beatrice
B
Macchi
D000480:Alkynes; D019380:Anti-HIV Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D048588:Benzoxazines / Q000494:pharmacology; D003521:Cyclopropanes; D018076:DNA, Complementary / Q000096:biosynthesis; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D019829:Nevirapine / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D048348:Reverse Transcription / Q000187:drug effects; D012680:Sensitivity and Specificity
D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D018076:DNA, Complementary; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; C098320:efavirenz
HIV-1 reverse transcriptase; antiviral agents; antiviral drug resistance; non-nucleoside reverse transcriptase inhibitor