Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

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

Which could be the relevance of constitutive N protein olimerization? RNA chaperones are nonspecific nucleic acid binding proteins with long disordered regions that help RNA molecules to adopt its functional conformation [50] [51] [52] . In agreement with this notion, which has already been proposed for CoV N proteins 40, 53 , our hypothesis is that recruitment of already formed N protein oligomers to the RTCs at DMVs and convoluted membranes via the interaction with nsp3 9, 10 , allows efficient and tight loading of the exceptionally large gRNA via numerous binding sites into a ribonucleoprotein complex (Fig. 5) . This RNA chaperone role of N protein oligomers would assure the efficient incorporation of the gRNA into the assembling virions, other scenarios, however, are also possible and future investigations will help to decipher the functional relevance of CoV N protein oligomerization. Plasmids. The sequences coding for either full-length MHV N protein or its truncations, i.e. N1 (amino acids 1 to 194), N2a (amino acids 195 to 257) and N2b-N3 (amino acids 258 to 454), were amplified by PCR from MHV gRNA and cloned into pET32c (EMD Millipore, Amsterdam, The Netherlands) and pGEX (GE Healthcare, Little Chalfont, United Kingdom) vectors using BamHI and XhoI, creating the pET32c-N, pET32C-N1, pET32C-N2a, pET32C-N2b-N3, pGEX-N, pGEX-N1, pGEX-N2a and pGEX-N2b-N3 constructs. The SARS-CoV N protein coding sequence or its truncations N1-N2a (amino acids 1 to 260) and N2a (amino acids 189 to 260) were also amplified by PCR and cloned into pET32c and pGEX vectors using XhoI and NotI to create pET32c-SARS-CoV-N, pET32c-SARS-CoV-N1-N2a, pGEX-SARS-CoV-N, pGEX-SARS-CoV-N1-N2a and pGEX-SARS-CoV-N2a.