Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein

Abstract

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus ( The assembly of coronaviruses is driven principally by homotypic and heterotypic interactions between the two most abundant virion proteins, the membrane protein (M) and the nucleocapsid protein (N) (14, 32). The M protein is a triplespanning transmembrane protein residing in the virion envelope, which is derived from the cellular budding site, the endoplasmic reticulum-Golgi intermediate compartment. More than half of the M molecule, its carboxy-terminal endodomain, is situated in the interior of the virion, where it contacts the nucleocapsid (46, 50). Also found in the virion envelope is the spike protein (S), which, although crucial for viral infectivity, is not an essential participant in assembly. The other canonical component of the coronavirus envelope is the small envelope protein (E), the function of which is enigmatic. Some evidence suggests that the E protein does not make sequence-specific contacts with other viral proteins (27) but instead functions by modifying the budding compartment, perhaps as an ion channel (56, 57). Alternatively, or additionally, E could act in a chaperone-like fashion to facilitate homotypic interactions between M protein monomers or oligomers (4).The N protein is the only protein constituent of the helically symmetric nucleocapsid, which is located in the interior of the virion. Coronavirus N proteins are largely basic phosphoproteins that share a moderate degree of homology across all three of the phylogenetic groups within the family (29). Some time ago, we proposed a model that pictured the N protein as comprising three domains separated by two spacers (A and B) (40). This arrangement was originally inferred from a sequence comparison of the N genes of multiple strains of the prototypical group 2 coronavirus, mouse hepatitis virus (MHV), and its validity seemed to be reinforced by numerous sequences that later became available. Part of this model, the delineation of spacer B and the acidic, carboxy-terminal domain 3, has been well supported by subsequent work (22,25,41,42). However, a wealth of recent, detailed structural studies of bacterially expressed domains of the N proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) and of infectious bronchitis virus (IBV) has much more precisely mapped boundaries within the remainder of the N molecule (8,16,21,23,47,51,60). The latter studies have shown that the N protein contains two independently folding domains, designated the N-terminal domain (NTD) and the C-terminal domain (CTD). It should be pointed out that this nomenclature can be misleading: the NTD does not contain the amino terminus of ...

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

“…S4). In addition, this upper shift of the N protein in SDS-PAGE were reported in a previous IBV N protein study (Yu et al, 2010) and other coronaviral N proteins (Hurst et al, 2009(Hurst et al, , 2010.…”

The Coronavirus Nucleocapsid Is a Multifunctional Protein

“…The charged LKR is also known as the SR-domain because it is rich in serine and arginine residues [34], and it is involved in cell signaling [15,35,36]. The flexible LKR is capable of direct interaction with RNA under in vitro conditions [37].…”

The SARS coronavirus nucleocapsid protein – Forms and functions

“…Second, the RNA-binding domain in the three-domain model could be re-defined to span both the NTD and the CTD. In fact, Hurst et al noticed that effective binding to RNA by MHV N protein in host cells required the presence of both the NTD and CTD (Hurst et al, 2009), suggesting that the NTD and CTD formed a single bipartite RNA interaction site, a feature to be reiterated in the final SARS-CoV RNP model. In this regard, the structure-based model is an evolution of the original three-domain model that provides a more refined framework for linking the structure and function of coronavirus N proteins.…”