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Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
Preferred sequences within a defined cleavage window specify DNA 3' end-directed cleavages by retroviral RNases H
Sharon J Schultz1, Miaohua Zhang, James J Champoux
1Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
The Journal of Biological Chemistry
|September 26, 2009
Summary
Reverse transcriptase RNase H activity involves three cleavage types. DNA 3' end-directed cleavages, like RNA 5' end-directed ones, depend on specific sequences within a narrow window, not just distance from the primer terminus.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Reverse transcriptase (RT) possesses RNase H activity crucial for retroviral replication.
- RNase H performs internal, RNA 5' end-directed, and DNA 3' end-directed cleavages.
- The role of sequence and distance in DNA 3' end-directed cleavage positioning was unclear.
Purpose of the Study:
- To investigate the determinants of DNA 3' end-directed cleavage by retroviral RNase H.
- To compare DNA 3' end-directed cleavage requirements with those of RNA 5' end-directed cleavage.
- To assess the impact of substrate sequence, distance, and end structure on cleavage site selection.
Main Methods:
- Utilized reverse transcriptases from human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV).
- Employed sequence-matched substrates to analyze cleavage patterns.
- Varied substrate end structure and primer terminus distance to observe effects on cleavage.
Main Results:
- DNA 3' end-directed cleavages exhibit similar sequence preferences to RNA 5' end-directed cleavages.
- Cleavage sites for DNA 3' end-directed activity are restricted to a narrow nucleotide window (15-20 for HIV-1 RT, 17-20 for M-MuLV RT).
- Specific RNA 5' end recessions accommodate both cleavage types, while further recession inhibits DNA 3' end-directed cleavage.
Conclusions:
- All three modes of retroviral RNase H cleavage share sequence determinants.
- The positioning of DNA 3' end-directed cleavage is governed by sequence preferences within a defined window.
- These findings can inform the design of novel assays for identifying retroviral RNase H inhibitors.
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