Evaluation of anti-HIV-1 mutagenic nucleoside analogues [Enzymology]

November 14th, 2014 by Vivet-Boudou, V., Isel, C., El Safadi, Y., Smyth, R. P., Laumond, G., Moog, C., Paillart, J. C., Marquet, R.

Due to their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of lethal mutagenesis that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base-pairs in DNA duplexes containing or not the nucleoside analogues: a promising candidate should display a small destabilisation of the matched base-pair compared to the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base-pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesised base pairs. Once again, only 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture since only compounds 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine were found to increase the mutation frequency, by 3.1 and 3.4 fold, respectively.