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Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase
O Hrusková-Heidingsfeldová1, M Andreansky, M Fábry
1Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague.
Abstract:
We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.
Insights
Researchers cloned the Mason-Pfizer monkey virus (MPMV) pro gene, revealing its self-processing activity. The resulting proteinase is enzymatically similar to myeloblastosis-associated virus proteinase, not HIV-1 proteinase.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- The Mason-Pfizer monkey virus (MPMV) is a retrovirus with a protease (pro) gene crucial for viral maturation.
- Understanding retroviral protease function is key to developing antiviral therapies.
Purpose of the Study:
- To clone and express the 3' region of the MPMV pro gene in Escherichia coli.
- To characterize the self-processing activity and enzymatic properties of the MPMV protease.
- To compare the MPMV protease's specificity to other retroviral proteases.
Main Methods:
- Cloning and expression of the MPMV pro gene in E. coli.
- In vitro self-processing assays of the recombinant precursor protein.
- Identification of NH2-terminal and COOH-terminal cleavage sites.
- Enzymatic activity and specificity assays using synthetic substrates.
- Inhibition studies with peptide inhibitors.
Main Results:
- The recombinant 26-kDa precursor protein rapidly self-processes to a 17-kDa active protein (p17) in E. coli and in vitro.
- Further in vitro processing yields active p12 and p5 fragments.
- The NH2-terminal processing site is similar to mouse mammary tumor virus proteinase.
- MPMV protease activity is more closely related to myeloblastosis-associated virus proteinase than to Human Immunodeficiency Virus type 1 proteinase.
- Inhibition studies support the enzymatic characterization.
Conclusions:
- The MPMV protease undergoes efficient self-processing to generate active forms.
- MPMV protease exhibits distinct enzymatic characteristics compared to HIV-1 protease.
- These findings provide insights into MPMV replication and potential therapeutic targets.