Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

Cong, Yingying; Kriegenburg, Franziska; de Haan, Cornelis A. M.; Reggiori, Fulvio

MHV N protein forms large oligomers in vitro. To gain insights into the self-assembly mechanism of the N protein, we decided to use an in vitro approach to exclude the eventual involvement of other factors in this event. Since order-disorder and secondary structure predictions coupled with sequence alignment has highlighted that all CoV N proteins have the modular organization as MHV N protein, we used this as a model first. Hence, MHV N protein was expressed in E. coli as a GST fusion construct and purified using glutathione Sepharose (GSH-beads). The immobilized GST fusion protein or GST was incubated with bacterial cell extract of E. coli expressing recombinant 6xHis-tagged full length MHV N protein. As illustrated in Fig. 1b , recombinant 6xHis-tagged N protein specifically bound GST-N protein but not GST alone, which is in agreement with previous reports 19, 23, 24, [27] [28] [29] and further shows that N proteins self-interact directly.