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Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein
1Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Abstract:
The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.
Insights
Researchers produced Moloney murine leukemia virus (M-MuLV) gene products in E. coli, discovering strong DNA-binding activity essential for provirus integration. This finding aids in understanding retroviral replication mechanisms.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The 3'-terminal region of the retroviral pol gene is crucial for integrated provirus formation post-infection.
- Understanding the function and properties of this pol gene product is vital for retroviral research.
Purpose of the Study:
- To express and isolate large quantities of the Moloney murine leukemia virus (M-MuLV) pol gene product.
- To characterize the DNA-binding activity of the expressed M-MuLV pol gene product.
Main Methods:
- Expressed portions of the M-MuLV pol gene as trpE fusion proteins in Escherichia coli.
- Utilized two methods for protein extraction and renaturation: polyacrylamide gel electrophoresis with nitrocellulose blotting and guanidine solubilization.
- Assayed DNA-binding activity of the renatured proteins.
Main Results:
- Successfully expressed M-MuLV pol gene fragments as fusion proteins in E. coli.
- The renatured proteins exhibited significant DNA-binding activity.
- Characterized the binding activity and compared it to authentic viral proteins.
Conclusions:
- The expressed M-MuLV pol gene products possess DNA-binding capabilities.
- These findings provide insights into the functional domains of the retroviral pol gene product involved in integration.
- The developed expression and assay methods facilitate further study of retroviral proteins.