Template properties of mutagenic cytosine analogues in reverse transcription

We have studied the mutagenic properties of ribonucleotide analogues by reverse trans-cription to understand their potential as antiretro-viral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(b-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribo-nucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse trans-criptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP oppo-site these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine ana-logues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome