Related Experiment Video
Updated: May 17, 2026
Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
Published on: December 21, 2019
Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules
Gary Ro-Lin Chang1, Min-Ying Wang, Jiahn-Haur Liao
1Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
Abstract:
Viral protein 4 (VP4) is a serine protease that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 of infectious bursal disease virus. In this report, the recombinant VP4 with a His-tag and three mutants (VP4-S652A, VP4-K692A and VP4-S652A.K692A) were expressed in Escherichia coli. Soluble VP4 was purified using immobilized metal-ion affinity chromatography or sucrose density gradient following with gel-filtration chromatography. The purified VP4 has a tubular structure with 25-30 nm in width and ∼300 nm in length, as observed by transmission electron microscope. A similar tubular structure was also found for these three mutants. The endopeptidase activity of these VP4 tubules was characterized by fluorescence resonance energy transfer using a synthetic fluorogenic oligopeptide as a substrate. The results show that the tubule-like VP4 is a functional enzyme with K(m) of 43 ± 2 μM and k(cat) of 0.04 ± 0.01 min⁻¹; however, k(cat) of three mutants were significantly reduced. This is the first report to demonstrate that VP4 protein expressed in E. coli can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni⁺² ions.
Insights
Infectious bursal disease virus protein 4 (VP4) self-assembles into functional tubules in E. coli. Mutants showed reduced protease activity, and Ni+2 ions inhibited VP4 completely.
Area of Science:
- Virology
- Structural Biology
- Biochemistry
Background:
- Infectious bursal disease virus (IBDV) poses a significant threat to poultry.
- Viral protein 4 (VP4) is a crucial serine protease involved in IBDV polyprotein processing.
- Understanding VP4 structure-function relationships is key to developing antiviral strategies.
Purpose of the Study:
- To express and purify recombinant VP4 and its mutants in E. coli.
- To characterize the self-assembly and enzymatic activity of VP4.
- To investigate the effect of mutations and metal ions on VP4 function.
Main Methods:
- Recombinant expression of VP4 and mutants in Escherichia coli.
- Purification using immobilized metal-ion affinity chromatography and gel-filtration.
- Transmission electron microscopy for structural analysis.
- Fluorescence resonance energy transfer assays for enzymatic activity.
Main Results:
- VP4 self-assembled into functional tubule-like particles in E. coli.
- Purified VP4 tubules exhibited endopeptidase activity (Km = 43 ± 2 μM, kcat = 0.04 ± 0.01 min⁻¹).
- Mutants displayed significantly reduced catalytic efficiency (kcat).
- Nickel ions (1 mM) completely inhibited VP4 activity.
Conclusions:
- This study demonstrates the self-assembly of functional VP4 tubules from E. coli.
- VP4's enzymatic activity is structure-dependent and sensitive to specific mutations.
- VP4 is a potential target for antiviral therapies, as evidenced by Ni+2 inhibition.
