Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

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

MHV N protein assembles in cytoplasmic oligomers over the course of an infection. We explored whether the N protein also forms oligomers over the course of an infection. In MHV-infected cells, the N protein localizes to three distinct locations: RTCs, virions and cytoplasm 2, 9, 10, 13, 42 . Since the N protein is in large complexes with other viral proteins in both RTCs and virions, we decided to focus on its cytoplasmic pool and to separate it from the two other populations through differential centrifugations. We generated cell extracts (Ext) from MHV-infected LR7 cells and centrifuged them at 15,000 × g to discard the pellet P13, which includes compartments with low sedimentation rates such as whole cells or debris, nuclei and mitochondria. The resulting supernatant S13 was subsequently centrifuged at 110,000 × g to sediment organelles such as the Golgi apparatus but also virions that could have remained associated to the cell surface at the moment of the lysis 43, 44 . Successful fractionation of each step was followed by visualization of the membrane bound protein VAPA for P13 45 as well as the cytoplasmic proteins GAPDH and tubulin for S45, which also contained soluble N protein (Fig. 2a) . Thus, we concluded that the cytoplasmic pool of the N protein is enriched in the S45 supernatant.