Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia...

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The human immunodeficiency virus type I (HIV-1) pol gene encodes essential viral enzymes, including reverse transcriptase and protease.
• Understanding the processing of the pol precursor polyprotein is crucial for developing antiviral strategies.

Purpose of the Study:

• To investigate the in vivo processing of the HIV-1 pol precursor polyprotein expressed in Escherichia coli.
• To characterize the enzymatic activities associated with the processed polypeptides.

Main Methods:

• Expression of the HIV-1 pol open reading frame in E. coli.
• Analysis of protein processing intermediates using molecular weight determination.
• Assay of reverse transcriptase activity in accumulated polypeptides.
• Site-directed mutagenesis of the putative protease active site.

Main Results:

• Protease-mediated processing of the pol precursor polyprotein was observed in E. coli.
• Two major polypeptides of 64 and 52 kilodaltons, exhibiting reverse transcriptase activity, accumulated.
• The HIV-1 protease moiety was processed into a 10-kilodalton species.
• A single amino acid substitution in the protease active site abolished polyprotein processing.

Conclusions:

• E. coli can support the correct processing of the HIV-1 pol polyprotein.
• The identified 64 and 52 kDa polypeptides are key products of pol precursor processing and harbor reverse transcriptase activity.
• The HIV-1 protease is essential for the efficient processing of the pol polyprotein.