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 meta-stable 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 5.2Å 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 (100-10Å) 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

Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo-electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the "spikes" around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.

Retinoschisin (RS1) is involved in cell-cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components.