RNase H Cleavage of tRNAProMediated by M-MuLV and HIV-1 Reverse Transcriptases

Abstract

RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNAPro 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 tRNAPro 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 tRNAPro primer is completely removed after plus-strand strong-stop synthesis.

Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection

“…PCR was used to detect circular viral DNA containing two long terminal repeats (LTRs). The primers used to amplify the LTR-LTR junction were MR5784 (5Ј-AGTCCTCCGATTGACTGAG-3Ј) and MR4091 (5Ј-CTCTTTTATTGAGCTCGGG-3Ј) (63). The PCR conditions were 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, repeated for 30 cycles.…”

Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition

“…7D). All three of the group 4 clones were single-base-pair insertions of T that were probably a result of cleavage 1 bp 5Ј from the end of the tRNA Pro primer (24,25). Three of the four group 2 clones revealed large deletions (Ͼ90 bp) in the 5Ј end of U3.…”

“…Three of these had an insertion of TG or TGG. The insertions of TG or TGG are most simply explained as a result of RNase H cleavage 2 or 3 bp 5Ј from the end of tRNA Pro during primer removal (24,25). Two of the viral insertion clones for group 4 included sequences from the PPT and are explained by plus-strand mispriming just upstream of the PPT or by the failure to remove the PPT primer.…”

“…LTR-LTR circle junction analysis was performed by a modification of a previously described protocol (25), which was utilized in this lab (1). Here primers ss-ps and U3-rev were used to amplify an R-U5-U3 region within circle junctions from low-molecular-weight DNA collected from acutely infected Rat2-2 cells.…”

Early Detection of a Two-Long-Terminal-Repeat Junction Molecule in the Cytoplasm of Recombinant Murine Leukemia Virus-Infected Cells

“…Retroviral linear DNA is the precursor to the integrated provirus (3,5,17), while inside the nucleus two other forms of retroviral DNA are traditionally described: covalently closed circular forms with one LTR (the product of recombination between the two LTRs of the linear DNA) or two tandems LTRs resulting either from blunt-end ligation of the duplex linear DNA, producing a circle junction often with CATT-AATG (43,44) between the LTR termini, or from autointegration, which produces significantly rearranged molecules. These circular DNA molecules were initially described in nuclei of avian sarcoma virus-infected cells (19,(41)(42)(43)49).…”