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An expanded model of replicating human immunodeficiency virus reverse transcriptase
B M Wöhrl1, C Tantillo, E Arnold
1Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Biochemistry
|April 25, 1995
Summary
Enzymatic footprinting reveals human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) encompasses more DNA substrate than previously thought. This suggests additional enzyme domains contribute to HIV-1 replication beyond the active centers.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is crucial for viral replication.
- Understanding the structural interactions of HIV-1 RT with its DNA substrate is key to developing antiviral therapies.
Purpose of the Study:
- To investigate the extent of DNA substrate binding by HIV-1 RT during replication.
- To reconcile structural and enzymatic data on HIV-1 RT-DNA interactions.
Main Methods:
- DNase I and S1 enzymatic footprinting assays were performed on replication complexes.
- Structural modeling of HIV-1 RT in complex with DNA was utilized.
Main Results:
- Enzymatic footprinting indicated a larger portion of the DNA primer-template is protected by HIV-1 RT compared to crystallographic data.
- Nucleotides from +7 to -23 of the template and -1 to -25 of the primer were nuclease resistant.
- A model incorporating an extended single-stranded template and duplex region was consistent with footprinting data.
Conclusions:
- HIV-1 RT engages a more extensive region of the DNA substrate than suggested by crystallography alone.
- Domains of HIV-1 RT proximal to the catalytic centers likely contribute to substrate binding and enzyme function.