Harnessing an RNA‐mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation

Abstract

A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.—Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

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, ).…”

“…The utility of chaperna system for the recombinant preparation of other pro-enzymes, including Tyr and TGF, merits further investigation. RNA-mediated protein-folding helper system has been proven superior to the classical fusion approaches in promoting solubility (Choi et al, 2009), and in the assembly of monomeric proteins into biologically relevant architecture (Y. S. S. W. Yang et al, 2018).…”

“…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, ).…”

Built‐in RNA‐mediated chaperone (chaperna) for antigen folding tailored to immunized hosts

“…The cell culture and lysate experiments were carried out based on the protocols described in Section 2.2 of Yang et al (2018). The E. coli total cell lysates (T) were centrifuged at 12,000 rpm for 10 min and separated into soluble (S) and pellet (P) fractions.…”

“…A novel protein‐folding function of RNA has recently been recognized; it remarkably outperforms protein‐based molecular chaperones in promoting protein folding and solubility (Choi, Ryu, & Seong, 2009; Horowitz & Bardwell, 2016; J. M. Kim, Choi, & Seong, 2017; Kwon, Yu, Park, Lee, & Seong, 2018; Kwon, Kim, et al, 2018; Son, Choi, Han, & Seong, 2015). This unique function has been recognized both in natural cellular environments and the host–virus interface during viral infections and could be extended to the expression of soluble recombinant proteins (Yang et al, 2018). A convenient avenue for harnessing an RNA‐dependent chaperone (chaperna: chaperone + RNA) is to fuse the target antigen of interest with an RNA‐interacting domain (RID) as a docking tag.…”