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Updated: May 5, 2026
Engineering Antiviral Agents via Surface Plasmon Resonance
Published on: June 14, 2022
Strategies for purifying variants of human rhinovirus 14 2C protein
Tomáš Sára1, Robert Konrat2, Tim Skern1
1Max F. Perutz Laboratories, Medical University of Vienna, Dept. of Medical Biochemistry, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.
Abstract:
The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.
Insights
Picornavirus 2C protein, crucial for RNA replication, shows significant variation across genera. This study models HRV14 2C and successfully expresses and purifies its domains, enabling future structural analysis of this key viral protein.
Area of Science:
- Virology
- Structural Biology
- Molecular Biology
Background:
- Picornaviruses utilize a positive-strand RNA genome translated into a polyprotein.
- The 2C protein, an ATPase and helicase, is essential for anchoring viral replication complexes to cellular membranes.
- The exact function of 2C remains unclear due to a lack of structural data, stemming from difficulties in producing soluble and stable protein.
Purpose of the Study:
- To investigate the heterogeneity of the 2C protein across different picornavirus genera.
- To develop computational models of human rhinovirus 14 (HRV14) 2C for future mutational analysis.
- To express and purify domains of HRV14 2C for structural and biophysical characterization.
Main Methods:
- Comparative analysis of 2C proteins from various picornavirus genera.
- Protein structure meta-analysis to generate homology models of HRV14 2C.
- Expression and purification of two HRV14 2C domains using three distinct protocols.
- Thermal denaturation and proton nuclear magnetic resonance ((1)H NMR) to assess protein folding and stability.
Main Results:
- Significant heterogeneity was observed in the 2C protein across different picornavirus genera.
- Computational models of HRV14 2C were successfully developed.
- Two domains of HRV14 2C were expressed and purified to sufficient concentrations for further structural studies.
- The purified domains showed characteristics amenable to crystallization and NMR analysis.
Conclusions:
- The study provides a foundational platform for exploring 2C proteins from all picornaviral genera.
- The developed HRV14 2C models and purified domains are valuable resources for future structural investigations.
- This work paves the way for detailed structural analysis of the 2C protein, potentially revealing its precise role in viral replication.
