Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requi ring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core partic le. Although the mechanism of assembly has been investigated extensively bo th in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis viru s nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-li nked species, a covalent dimer, has been detected only in the presence of n ucleic acid and with capsid proteins capable of binding nucleic acid. Optim um nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-ac id ratio identical to that required for maximum binding of the capsid prote in to nucleic acid. Identical results were observed when cross-linking in v itro assembled core particles of both Sindbis and Ross River viruses. Purif ied cross-linked dimers of truncated proteins and of mutant proteins that f ailed to assemble were found to incorporate into assembled core particles w hen present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly , Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in r elation to the existing model of the nucleocapsid core suggests that the cr oss-linked species is a cross-capsomere contact between a pentamer and hexa mer at the quasi-threefold axis or is a cross-capsomere contact between hex amers at the threefold axis of the icosahedral core particle and suggests s everal possible assembly models involving a nucleic acid-bound dimer of cap sid protein as an early step in the assembly pathway.