Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium towards particle formation by promoting inter-subunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in E. coli, the delta domain guides 420 copies of the subunit into a procapsid with T=7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it 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. Prohead-I+P was isolated by co-expressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I+P was determined at 5.2Å resolution and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I+P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.
展开▼
