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The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase
K A Pullen1, A J Rattray, J J Champoux
1Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Abstract:
A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.
Insights
Researchers investigated how reverse transcriptase enzymes initiate DNA synthesis. They found that while both Moloney murine leukemia virus (M-MuLV) and human immunodeficiency virus type 1 (HIV-1) use similar polypurine tracts (PPTs), the specific DNA sequences critical for priming differ between the two viruses.
Area of Science:
- Molecular Biology
- Virology
- Enzymology
Background:
- Reverse transcriptase enzymes are crucial for viral replication, synthesizing DNA from RNA templates.
- The RNase H activity of reverse transcriptase cleaves RNA primers, initiating plus-strand DNA synthesis.
- The polypurine tract (PPT) is a key sequence element directing this cleavage and priming event.
Purpose of the Study:
- To investigate the sequence requirements for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase-mediated plus-strand DNA priming at the polypurine tract (PPT).
- To compare the priming specificity of HIV-1 reverse transcriptase with that of Moloney murine leukemia virus (M-MuLV) reverse transcriptase.
- To identify critical nucleotide residues within the PPT that dictate RNase H cleavage site selection for HIV-1.
Main Methods:
- Utilized site-directed mutagenesis to generate a series of polypurine tract (PPT) mutants in both Moloney murine leukemia virus (M-MuLV) and human immunodeficiency virus type 1 (HIV-1).
- Assessed the ability of HIV-1 reverse transcriptase to initiate plus-strand DNA synthesis using M-MuLV PPT sequences in vitro.
- Analyzed the specificity of RNase H-mediated cleavage at the PPT in the presence of HIV-1 reverse transcriptase and various PPT mutants.
Main Results:
- HIV-1 reverse transcriptase can utilize the M-MuLV PPT as an origin for plus-strand DNA initiation in vitro.
- Despite sequence similarities in the PPT region between M-MuLV and HIV-1, the critical determinants for RNase H cleavage and primer generation differ significantly.
- For HIV-1, the -2G and -4G residues within the PPT are key determinants of priming specificity, contrasting with the -7A residue critical for M-MuLV.
Conclusions:
- The sequence features governing RNase H positioning and cleavage for plus-strand priming are virus-specific, even for closely related functional elements like the PPT.
- Understanding these sequence specificities is vital for comprehending viral replication mechanisms and for developing targeted antiviral therapies.
- The study highlights distinct molecular recognition mechanisms employed by different retroviral reverse transcriptases at the polypurine tract.