Chaperna-Mediated Assembly of Ferritin-Based Middle East Respiratory Syndrome-Coronavirus Nanoparticles

Abstract

The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.

Retrospective analysis of laboratory testing in 54 patients with severe- or critical-type 2019 novel coronavirus pneumonia

“…We believe that the SARS-CoV-2 infection caused the increases in serum ferritin, which did not change as patients improved, and therefore cannot be used for disease assessment. However, another study [4] has determined that assembly of Middle East Respiratory Syndrome (MERS) coronavirus nanoparticles is related to a chaperone-mediated ferritin. The specific relationship between ferritin and coronavirus infection in humans therefore requires further study.…”

RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

“…RNA‐mediated protein‐folding helper system has been proven superior to the classical fusion approaches in promoting solubility (Choi et al, ), and in the assembly of monomeric proteins into biologically relevant architecture (Y. S. Kim et al, ; S. W. Yang et al, ). Although the understanding the detailed mechanism warrants further studies, it is plausible that the polyanionic backbone of RNA with electrostatic repulsion and steric hindrance may desist intermolecular aggregation of folding intermediates into nonfunctional form, favoring productive folding (Choi et al, ; Zhang et al, ).…”

“…These are distinct from the RNA chaperone, which is a protein that facilitates structural alteration of RNA molecules. In an engineered system, the chaperna could be functionally harnessed to fold and assemble recombinant proteins into immunologically relevant conformations (Y. S. Kim et al, ; S. W. Yang et al, ).…”

Molecular Characteristics, Functions, and Related Pathogenicity of MERS-CoV Proteins

“…There is no completely effective treatment for MERS-CoV. Current treatment methods mainly imitate the treatment of SARS-CoV, and comprise the following: symptomatic treatment, including biological therapy such as monoclonal antibodies (mAbs), blockers, protease inhibitors, corticosteroids, and IFN; chemical drugs, such as Acyclovir [61], chlorpromazine hydrochloride [62], loperamide, and lopinavir [63]; broad-spectrum antibiotics, such as ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [64]; and nanoparticle vaccines [65]. However, wellorganized clinical trials have not yet been carried out.…”