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Updated: Aug 8, 2026
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Published on: March 22, 2016
Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites
1Department of Microbiology and Immunology, University of California, San Francisco 94143.
Abstract:
The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.
Insights
Moloney murine leukemia virus integration protein (IN) specifically binds att sites on viral DNA. Key sequences, including the CA dinucleotide, are essential for this DNA binding activity.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The integration protein (IN) is crucial for retroviral DNA integration.
- Moloney murine leukemia virus (MuLV) IN mediates the integration of viral DNA into the host genome.
- Understanding IN's DNA binding is key to deciphering retroviral replication.
Purpose of the Study:
- To analyze the binding of Moloney MuLV IN to its att sites.
- To define the specific sequence requirements for IN-MuLV att site recognition.
- To establish a functional assay for IN's sequence-specific DNA binding activity.
Main Methods:
- Purification of Moloney MuLV IN from yeast cells.
- Electrophoretic mobility shift assay (EMSA) using synthetic oligonucleotides containing att sequences.
- Site-directed mutagenesis of att sequences to assess binding affinity.
Main Results:
- Moloney MuLV IN specifically binds to MuLV att sequences in a sequence-dependent manner.
- The terminal 13 base pairs of att sites are sufficient for binding when oriented correctly.
- The conserved CA dinucleotide preceding the att site is critical for IN binding; mutations significantly reduce or abolish binding.
- Binding is dependent on the presence of the A residue immediately upstream of the 3' processing site.
Conclusions:
- The study defines critical sequence elements within att sites required for Moloney MuLV IN recognition and binding.
- The conserved CA dinucleotide plays a vital role in att site binding, explaining its evolutionary conservation.
- The findings provide a foundation for understanding IN-mediated integration and offer a tool for assessing IN activity.
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