The SARS-CoV-2 Nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Abstract

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

“…The solution environment and types of RNA used in our experiments are very different from the cytoplasm and viral RNA. However, similar results have been obtained in published and unpublished work by several other groups under a variety of conditions, including via in cell experiments [20][21][22][23][24][25][26][27] . Taken together, these results demonstrate that N protein can undergo bona fide phase separation, and that N protein condensates can form in cells.…”

“…Finally, given we observed phase separation with poly(rU), the behavior we are observing is likely driven by relatively nonspecific protein:RNA interactions. In agreement, work from a number of other groups has also established this phenomenon across a range of solution conditions and RNA types [20][21][22][23][24][25][26][27] .…”

“…Recent hydrogen-deuterium exchange mass spectrometry also identified H6 43 . Residues in this region have previously been identified as mediating M-protein binding in other coronaviruses, such that we propose H6 underlies that interaction 21,[104][105][106] . Recent work has also identified amphipathic transient helices in disordered proteins as interacting directly with membranes, such that an additional (albeit entirely speculative) role could involve direct membrane interaction, as has been observed in other viral phosphoproteins 107,108 .…”

G-Quadruplexes in RNA Biology: Recent Advances and Future Directions

“…This mechanism was also reported for the nsp3 of SARS coronavirus-severe acute respiratory syndrome coronavirus (SARS-CoV) [90] and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [91]. The interaction between RG4s and viral proteins might also contribute to LLPS-dependent viral RNA packaging and host proteins co-opting [92][93][94].…”

Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress

“…M, E and S are membrane-associated proteins and are localized to the ER, Golgi and the ERGIC 30 , 44 . The N protein associates with the genomic vRNA and M protein, which presumably drives vRNA packaging and genome encapsidation 45 , 46 . The main assembly and budding site of other coronaviruses has been previously described at the ERGIC by conventional EM of stained plastic sections 7 , 28 , 30 , 44 .…”