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Plus-strand priming by Moloney murine leukemia virus. The sequence features important for cleavage by RNase H
1Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Abstract:
The reverse transcriptase-associated RNase H activity is responsible for producing the plus-strand RNA primer during reverse transcription. The major plus-strand initiation site is located within a highly conserved polypurine tract (PPT), and initiation of DNA replication at this site is necessary for proper formation of the viral long terminal repeats (LTRs). We present here a compilation of PPT sequences from an evolutionarily diverse group of retroviruses and retrotransposons, which reveals that there is a high degree of sequence conservation at this site. Furthermore, we found previously that secondary plus-strand origins, identified in vitro, also show strong similarity to the PPT. Taken together, these data suggest that RNase H recognizes a specific sequence at the PPT as a signal to cleave the RNA at a precise location, producing a primer for the initiation of plus-DNA strands. We have analyzed the RNase H recognition sequence by producing a large number of single and double mutations within the PPT. Our findings suggest that no single residue in the +5 to -6 region (where the cleavage occurs between -1 and +1) is essential; mutations at these positions introduced heterogeneity at the cleavage site, but cleavage is still predominantly at the correct location. Furthermore, base-pairing is not required at the +1 position of the RNase H cleavage site, but a mismatched base-pair at the -1 position causes imprecision in the cleavage reaction. Interestingly, the A residue at position -7 seems to be critical in positioning the RNase H enzyme for correct cleavage. The preference of the enzyme for cleaving between G and A residues may play a minor role in determining the specificity.
Insights
The polypurine tract (PPT) sequence is crucial for reverse transcriptase-associated RNase H to initiate DNA replication. Specific sequence elements within the PPT guide precise RNA cleavage, ensuring correct primer formation for viral DNA synthesis.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Reverse transcriptase (RT) in retroviruses and retrotransposons possesses RNase H activity essential for viral replication.
- This activity generates the plus-strand DNA primer by cleaving the RNA template at a specific site.
- The major initiation site for this process is the highly conserved polypurine tract (PPT).
Purpose of the Study:
- To investigate the sequence requirements of the polypurine tract (PPT) for RNase H-mediated RNA cleavage.
- To identify critical residues and structural features within the PPT that dictate precise primer generation.
- To understand how RNase H recognizes and binds to the PPT for accurate cleavage.
Main Methods:
- Compilation and analysis of PPT sequences from diverse retroviruses and retrotransposons.
- Site-directed mutagenesis of the PPT sequence to generate single and double mutants.
- Assessment of RNase H cleavage site specificity in vitro using mutated PPT sequences.
Main Results:
- High sequence conservation observed in the PPT across various retroelements, suggesting functional importance.
- Mutations within the +5 to -6 region of the PPT did not abolish cleavage but introduced heterogeneity.
- Base-pairing at the +1 position was not essential, but a mismatch at -1 caused imprecise cleavage.
- The adenine residue at position -7 was identified as critical for accurate RNase H positioning and cleavage.
Conclusions:
- The PPT sequence serves as a critical recognition signal for RNase H, ensuring precise initiation of plus-strand DNA synthesis.
- Specific sequence features, particularly the A at -7, are vital for guiding RNase H activity.
- Understanding these interactions advances knowledge of retroviral replication mechanisms and potential therapeutic targets.