Representative Examples of Viral Assembly in Heterologous Expression Systems

Representative Examples of Viral Assembly in Heterologous Expression Systems 

Truncated versions of the nucleocapsid protein that lacked various portions of the N terminus revealed that deletion of the first 32 amino acids interfered with assembly, but this deletion did not eliminate interaction of the coat protein with nucleic acid. When present in small amounts, this mutant coat protein could be incorporated into capsids assembled from the wild-type protein. However, when present in large amounts, the mutant coat protein inhibited assembly of the wild-type protein. 

The observation that nucleocapsid assembly in the absence of nucleic acids was inhibited indicated that interaction of the coat protein with the RNA is an essential and early step in the assembly pathway. The availability of mutant coat proteins that retained nucleic acid-binding activity but could not assemble further provided an opportunity to identify a possible coat protein-nucleic acid assembly intermediate. Cross-linking experiments revealed the presence of a coat protein dimer that could be detected only in the presence of nucleic acid and for those types of mutant proteins that had retained nucleic acid-binding activity. The protein dimer itself could not assemble into cores but was incorporated into cores in the presence of wild-type protein. These and other results strongly suggested that the cross-linked dimer represents a genuine intermediate of nucleocapsid core assembly. 

Further analysis of the cross-linked intermediate showed that lysine at position 250 of one capsid subunit was covalently linked to the identical amino acid on a second subunit. In a model of the nucleocapsid derived from both cryoelectron microscopy (cryo-EM) analysis and X-ray analysis of the nucleocapsid protein, the covalent bond connects a pentamer of coat proteins with a hexamer of coat proteins, that is, it is an intercapsomer contact rather than an intracapsomer contact. This finding was unexpected because a possible assembly model proposed preassembly of pentameric and hexameric units that would recruit RNA for assembly into the final structure. In light of the new data, however, this scenario is unlikely. Instead, the initial assembly intermediate appears to be a coat protein dimer bound to RNA and the dimer spans the intercapsomere space. 

Polyoma- and papillomaviruses share a common capsid structure that is assembled from 72 pentameric capsomeres arranged on a T = 7 icosahedral lattice. Assembly studies of polyomaviruses were first initiated by expression of the major capsid protein of mouse polyomavirus, VPl, in 

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