Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• An expression cassette utilizing tobacco etch virus (TEV) NIa protease was previously developed for coordinated protein accumulation via polyprotein self-processing.
• Low protein yields were observed when the full-length NIa protease was part of the polyprotein.

Purpose of the Study:

• To investigate if disrupting the nuclear localization of TEV NIa protease affects protein levels produced by the expression cassette.
• To optimize protein accumulation by modifying NIa protease localization signals.

Main Methods:

• Modified TEV NIa protease by removing nuclear localization signals (NLSs) or the VPg domain, or by fusing it to a cytoplasmic anchor protein.
• Evaluated modified NIa protease activity using in vitro translation and in vivo protoplast experiments.
• Assessed protein accumulation using GUS reporter assays and analysis of viral coat protein (CP) synthesis.

Main Results:

• Modified NIa protease retained sequence-specific proteolytic activity.
• Removal of NLSs led to increased GUS reporter accumulation.
• The pPRO10 cassette facilitated the synthesis of NIa and up to three viral CPs.
• Protein accumulation in protoplasts was dependent on CP coding sequence position and protein stability.

Conclusions:

• Disrupting NIa protease nuclear localization enhances protein accumulation in the expression cassette.
• The modified cassette, pPRO10, shows potential for producing multiple viral proteins.
• Protein accumulation is influenced by cassette design and protein stability, requiring further optimization.