Abstract
Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.
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Acknowledgements
The authors are indebted to IRCCS Centro Neurolesi Bonino-Pulejo, Messina, Italy (FMM scholarship), and to the Institute of Translational Pharmacology, CNR, Rome, Italy (CF and EP scholarships). This work was supported by the Italian Ministry of University and Research, Projects of National Interest (PRIN, to B. Macchi), by the Institute of Translational Pharmacology (A. Mastino) and by the IRCCS Centro Neurolesi Bonino-Pulejo, Messina (F. Marino-Merlo).
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Marino-Merlo, F., Frezza, C., Papaianni, E. et al. Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase. Appl Microbiol Biotechnol 101, 8249–8258 (2017). https://doi.org/10.1007/s00253-017-8544-6
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DOI: https://doi.org/10.1007/s00253-017-8544-6