Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

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

Next we explored whether there was any putative redundancy in binding between the different domains of the N protein to mediate N protein oligomerization. For this, bacterial cell extracts from E. coli expressing 6xHis-tagged N1, N2a or N2b-N3 were incubated with immobilized GST or GST-N protein as well as the truncations GST-N1, GST-N2a and GST-N2b-N3. Interestingly, all constructs were able to specifically bind both the N1 and N2b-N3 truncations suggesting that each domain within the N protein could interact with at least two different regions from another N protein molecule (Fig. 3) . Further, 6xHis-tagged N1 and 6xHis-tagged N2b-N3 displayed a binding with immobilized GST-N2a. However, there was no binding between 6xHis-tagged N2a and immobilized GST-N2a indicating that the N2a domain might participate to the oligomer formation without being one of the critical determinants. 6xHis-tagged N2b-N3 was pulled down by GST-tagged N1 protein irrespectively of RNase A treatment confirming that the studied bindings do not depend on bacterial RNA ( Supplementary Fig. S1d ).