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Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery

Significance Particle instability, antigen fusion limitations, and intrinsic immunogenicity greatly limit the development of virus-like particles (VLPs). To address these problems, the Hepatitis B core protein (HBc) VLP was extensively modified to improve its functional properties by introducing an artificial disulfide network, removing the surface charge, and transplanting a new surface spike region. This study systematically explores the plasticity of viral capsids and, thereby, expands our knowledge regarding the relevant structure function relationships. Notably, we uncovered the influence of the HBc spike and its negative charges on capsid assembly and immunogenicity. Our observations on the unexpected importance of the HBc spike tip charged state suggest unexplored mechanisms for antiviral therapeutics that block capsid assembly. Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.