In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of th e glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature viri on. The nucleocapsid proteins of Sindbis virus and Ross River virus have be en produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, th e proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles . Truncated forms of the Sindbis capsid protein were used to establish amin o acid requirements for assembly. A capsid protein starting at residue 19 [ CP(19-264)] was fully competent for in vitro assembly, whereas proteins wit h further N-terminal truncations could not support assembly. However, a cap sid protein starting at residue 32 or 81 was able to incorporate into parti cles in the presence of CP(19-264) or could inhibit assembly if its molar r atio relative to CB(19-264) was greater than 1:1. This system provides a ba sis for the molecular dissection of alphavirus core assembly.