RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNA(Pro) is removed were constructed. This substrate was used to assay the ability of M-MuLV reverse transcriptase (RT) to cleave the RNA portion of the substrate. The cleavage specificities of the cognate M-MuLV RT and the heterologous enzyme from the human immunodeficiency virus type-1 (HIV-1) were compared. M-MuLV and HIV-1 RT recognize and cleave the RNA at distinct positions. The site of the initial RNase H cleavage in vitro was determined using 3' end nearest neighbor analysis of the initial cleavage product. M-MuLV RT/RNase H removed the model tRNA(Pro) between the terminal ribo-A and ribo-C, resulting in a terminal ribo-A attached to the viral DNA, whereas HIV-1 RT/RNase H was shown to cleave at the RNA-DNA junction. Analysis of the DNA over time indicated that the ribo-A is subsequently removed by M-MuLV RT. In vivo analysis from double-LTR circle junctions illustrated that 16 of the 23 clones isolated possessed the predicted junction if complete removal of the tRNA primer were to occur. The predicted junction for complete removal of the tRNA primer was CATT-AATG. One aberrant circle junction was isolated which could result from the use of an alternative primer. In contrast with HIV, no M-MuLV circle junctions were isolated which indicated processing of a single-LTR terminus by integrase. Analysis from in vivo and in vitro studies indicate that the M-MuLV tRNA(Pro) primer is completely removed after plus-strand strong-stop synthesis.
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