Effect of uracil incorporation on the precision of HIV-1 (+)-strand DNA initiation

Retroviruses have means to limit the accumulation of uracil in DNA strands, suggesting that uracil affects one or more steps in the viral life cycle. In this work, the effects of uracilated DNA were investigated in biochemical assays of (-)-strand DNA synthesis, RNase H activity and (+)-strand synthesis in a cell-free system that uses the activities of HIV-1 reverse transcriptase (RT) to covert single-stand RNA to double-stand DNA. Substitution of dUTP for dTTP did not alter (-)-strand synthesis but significantly decreased (+)-strand synthesis. Mapping of nascent (+)-strand DNA 5' ends revealed that this was due to reduced initiation from polypurine tracts (PPTs) with a concomitant increase in initiation at non-PPT sites. Aberrant initiation correlated with imprecise RNase H cleavage of RNA when assayed on preformed RNA-DNA duplexes containing uracilated DNA. Thus thymine C5 methyl groups influence RNase H specificity. In light of these data, a model is proposed to explain why retroviruses have mechanisma to limit uracil accumulation in viral DNA.