Structural studies of bacteriophage Hong Kong 97 prohead I and development of nano-particle applications.

摘要

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to correctly form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to promote subunit interactions and to stabilize assembly intermediates. HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain directing 420 subunits to form a procapsid named Prohead I. Prohead I can be disassembled and reassembled under mild conditions but cannot mature further. When the virally encoded protease (gp4) is co-expressed with gp5, it is incorporated into the capsid and digests the delta domain followed by auto-proteolysis to produce the metastable Prohead II that matures through multiple intermediates to form the polyhedral Head II capsid. A version of Prohead I (Prohead I+P) was isolated by co-expressing gp5 and an enzymatically inactive mutant of gp4. Prohead I and Prohead I+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid from disassembly in vitro. The crystal structure of Prohead I+P was determined at 5.2A and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead II. Prohead I differed from Prohead II due to the presence of the delta domain and the resulting repositioning of residues 104-130, explaining why Prohead I can be reversibly dissociated but cannot mature. Low-resolution x-ray crystal diffraction data (100-10A) enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.;Next-generation nanodevices are under development for delivering therapeutic agents to specific tissues more effectively than conventional strategies to reduce toxicity and side effects. HK97 capsid system was employed to utilize its multi-valency and stability for this purpose. We developed a method of packaging various fluorescent proteins by expressing them as fusion proteins with the HK97 protease. We observed that mCherry-protease fusion protein retains its enzymatic activity after packaging. Thus, the mCherry-containing capsid can be matured, producing a fully cross-linked particle carrying specific cargo. Furthermore, we engineered HK97 capsid for tumor cell-specific targeting. A combination of genetic and chemical engineering methods were developed and applied to generate dual-labeled particles displaying transferrin and fluorescent labels. The targeting properties of transferrin-conjugated particles were evaluated in in vitro experiments using different cancer cell lines. We found that HK97-tranferrin formulations were effectively targeted to cancer cells in vitro via the transferrin receptor. These studies highlight the utility and facilitate the further development of HK97-based VNPs for targeted protein delivery.