Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The human T-cell lymphotropic virus (HTLV) possesses key enzymes like protease and reverse transcriptase essential for its replication.
• Efficient expression systems are crucial for studying viral enzyme functions and developing antiviral strategies.

Purpose of the Study:

• To establish a high-level expression system for HTLV pol gene products in Bacillus subtilis.
• To characterize the expression and activity of HTLV protease and reverse transcriptase in a prokaryotic host.

Main Methods:

• A 2.4-kb DNA fragment encoding HTLV protease, reverse transcriptase, and N-terminal endonuclease was cloned into a Bacillus subtilis expression vector (pRTL11).
• Expression was placed under inducible control of Escherichia coli lac regulatory elements, utilizing IPTG induction.
• The resulting polypeptides were analyzed for size and enzymatic activity after partial purification.

Main Results:

• A 90-kDa polyprotein precursor was synthesized upon induction.
• Proteolytic cleavage yielded 64-kDa and 52-kDa polypeptides.
• Reverse transcriptase activity was detected and co-migrated with these two cleaved polypeptides.

Conclusions:

• Bacillus subtilis can be utilized as a host for the high-level expression of functional HTLV protease and reverse transcriptase.
• The study demonstrates the successful production and partial characterization of active HTLV enzymes in a prokaryotic system.