Analytical Biochemistry 2008-03-01

Monitoring the development of non-nucleoside reverse transcriptase inhibitor-associated resistant HIV-1 using an electrochemiluminescence-based reverse transcriptase polymerase assay.

Vandna Munshi, Meiqing Lu, Peter Felock, Richard J O Barnard, Daria J Hazuda, Michael D Miller, Ming-Tain Lai

Index: Anal. Biochem. 374(1) , 121-32, (2008)

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Abstract

Reverse transcriptase (RT) plays an essential role in the HIV-1 replication process, which converts a single-strand RNA into a double-strand DNA via polymerase and RNase H activities. Therefore, inhibition of RT has been one of the primary therapeutic strategies for suppressing the replication of HIV-1. To facilitate the process of discovering the next generation of antiretroviral agents, this study presents a highly sensitive and nonradioactive RT polymerase assay that is based on electrochemiluminescence (ECL) technology, where a ruthenylated dUTP (Ru-dUTP) is employed as one of the dNTPs. The concentration of the RT enzymes required for the assay can be as low as 1 pM, enabling us to evaluate inhibitors with low picomolar potency. More importantly, the assay is capable of detecting endogenous RT activity in cell-free viruses. Therefore, the assay was applied to monitor the development of resistance mutation(s) by viruses under the treatment with a non-nucleoside reverse transcriptase inhibitor (NNRTI) in cell culture. The magnitude of resistance of the resulting mutant viruses was assessed directly by the assay, eliminating the need for cloning, expressing, and purifying the RT mutants.