Analysis of the proteolytic processing and activation of the rice tungro bacilliform virus reverse transcriptase

Area of Science:

• Plant virology
• Molecular biology
• Protein biochemistry

Background:

• Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus.
• Open reading frame (ORF) 3 encodes viral capsid protein, protease (PR), and reverse transcriptase (RT).
• Previous expression of ORF 3 in insect cells yielded processed p62 and p55 proteins with RT activity.

Purpose of the Study:

• To determine the precise molecular weights and C-termini of p62 and p55 proteins using mass spectrometry.
• To express and purify recombinant p62R and p55R proteins and confirm their enzymatic activities.
• To investigate the role of the RTBV PR in processing the RT polyprotein by mutating its active site.

Main Methods:

• Expression of RTBV ORF 3 fragments and mutated ORFs in insect cells using baculoviruses.
• Purification of recombinant p62R and p55R proteins.
• Mass spectrometry for protein characterization.
• Site-directed mutagenesis of the putative PR active site.

Main Results:

• Mass spectrometry confirmed the molecular weights and C-termini of p62 and p55.
• Recombinant p62R and p55R proteins exhibited reverse transcriptase activity.
• Mutation of the PR active site (mpr/RT) resulted in an unprocessed ~87-kDa polyprotein in insect cells.

Conclusions:

• The RTBV PR is essential for the processing of the RT polyprotein.
• The study provides further understanding of the p62 and p55 proteins and their functions.
• This research clarifies the specific role of the RTBV PR in polyprotein maturation.