Analysis of polypurine tract-associated DNA plus-strand priming in vivo utilizing a plant pararetroviral vector carrying redundant ectopic priming elements

Initiation of DNA plus-strand synthesis in most reverse-transcribing elemen ts requires primer generation by reverse transcriptase-associated RNase H a t one or more template polypurine tracts (PPTs). We have exploited infectio us clones of the plant pararetrovirus cauliflower mosaic virus carrying red undant ectopic plus-strand priming elements to study priming in vivo. Ectop ic priming generated an additional discontinuity in progeny virion DNA duri ng infection of plants. We found that altering the length of the 13-base pa ir PPT by +/-25% significantly reduced priming efficiency. A short pyrimidi ne tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type pri mer. Use of an ectopic redundant primer to study replication-dependent prim ing was validated by demonstrating that it could rescue infectivity followi ng destruction of the wild-type priming elements. We propose a model for pl ant pararetroviral plus-strand priming in which pyrimidines enhance PPT rec ognition during polymerase-dependent RNase H cleavages, and suggest that fi delity of primer maturation during polymerase-independent cleavages involve s PPT length measurement and 3' end recognition by RNase H.