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Updated: May 10, 2026
Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly
Published on: March 1, 2012
Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells
Maria Gullberg1, Bartosz Muszynski1, Lindsey J Organtini2
1National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
Abstract:
The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C(pro)) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C(pro) can be toxic for cells. The expression level of 3C(pro) activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C(pro) were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C(pro) expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C(pro) with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvβ6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.
Insights
Controlling foot-and-mouth disease virus (FMDV) 3C protease activity improves capsid protein production. Modified expression systems yield higher amounts of processed FMDV capsid proteins that self-assemble into empty particles.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Foot-and-mouth disease virus (FMDV) structural protein precursor P1-2A processing relies on viral 3C protease (3C(pro)).
- High 3C(pro) expression can be toxic to cells, limiting production of FMDV capsid proteins.
- Optimizing 3C(pro) activity relative to P1-2A is crucial for efficient capsid protein yield and assembly.
Purpose of the Study:
- To investigate methods for reducing 3C(pro) toxicity while enhancing FMDV capsid protein production.
- To determine the impact of modulated 3C(pro) expression on the yield and assembly of FMDV empty capsid particles in mammalian cells.
- To analyze the morphology and receptor-binding properties of self-assembled FMDV empty capsids.
Main Methods:
- Utilized a vaccinia virus-based transient expression system to express FMDV P1-2A and 3C(pro) from monocistronic and dicistronic cassettes.
- Engineered constructs with reduced 3C(pro) catalytic activity and utilized mutant internal ribosome entry site (IRES) elements (GAGA and GTTA) for controlled 3C(pro) expression.
- Analyzed protein yield, self-assembly into empty capsids via electron microscopy and reconstruction, and receptor binding to integrin αvβ6.
Main Results:
- The P1-2A-mIRES-3C construct with the inefficient GTTA mutant IRES yielded the highest amount of processed capsid proteins for both FMDV serotypes O and A.
- These processed proteins self-assembled into FMDV empty capsid particles with distinct morphologies compared to X-ray crystallography data.
- The assembled empty capsids demonstrated divalent cation-dependent binding to the integrin αvβ6 receptor and were recognized in serotype-specific ELISAs.
Conclusions:
- Modulating 3C(pro) expression using mutant IRES elements is an effective strategy to overcome toxicity and increase FMDV capsid protein production.
- The self-assembled empty capsids are structurally relevant and retain functional binding to the cellular receptor αvβ6.
- This approach offers a promising avenue for the production of FMDV antigens for diagnostic and potential vaccine development.
