Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

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

protein dimers are the basic building blocks of the ribonucleoprotein complex 12, 23, 24, [27] [28] [29] . Subsequently, native gel electrophoresis, size exclusion chromatography and surface plasmon resonance revealed that recombinant SARS-CoV N protein forms oligomers in vitro, although it appears to predominantly exist as a dimer in solution in absence of gRNA 30 . Size exclusion chromatography and chemical cross-linking assays were also used to unveil that the N2b/CTD domain of the SARS-CoV N protein, in particular the stretch of amino acids between positions 343-402, forms autonomously oligomers in solution 31, 32 . The N2b/CTD domain of the MHV N protein has also been shown to bind full length protein 11 . However, few other studies indicated that other parts of the N protein could also be involved in the self-interaction. In particular, the N1b/NTD and N3 domains could bind full length MHV N protein 11, 19 while the serine-rich (SR) region located within the N2a could be essential for SAR-CoV N protein self-interaction and oligomerization 33 . Nonetheless, the current most commonly accepted working model is that CoV N protein constitutively dimerizes, primarily via the N2b/CTD domain, and subsequently oligomerizes during virion assembly through a mechanism that remains unclear 22, 23, 27, 29, 32, [34] [35] [36] . It is also unclear whether gRNA binding influences CoV N protein oligomerization. In this study, we confirm that recombinant MHV and SARS-CoV N protein self-interact to form large oligomers. However, we could also show that, besides N2b/CTD, several domains of the N protein are involved in this process. Analysis of different MHV and SARS-CoV N protein truncations revealed that at least three regions of these proteins cross-interact between each other in an interchangeable manner. Moreover, we show that two of these regions, i.e. N1 and N2b-N3, can oligomerize autonomously. Further, in infected cells, the MHV N protein forms oligomers already in the cytoplasm and its oligomerization does not require binding to gRNA. Altogether these findings indicate that CoV N proteins self-interact and oligomerize via discontinuous regions present in domains distributed over the entire protein to generate large supra-complexes. We hypothesize that these oligomers, which are formed constitutively, provide a larger binding surface for the gRNA, which will be thus optimally engaged at the RTCs and subsequently incorporated into forming viral particles.