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N protein for the N protein before 2000, namely the TRS (Stohlman et al.,
Residing within the virion interior, N protein is the sole protein 1988) and the genomic packaging signal (Molenkamp and Spaan,
constituent of the helical nucleocapsid (Fung and Liu, 2018). 1997), the N protein is also known to bind to non-structural
Monomers of this 43–50 kDa protein bind the RNA genome protein 3 (nsp3) and M protein to help tether the viral genome
in a ‘beads on a string’ configuration. Crystal structures of IBV into the replicase-transcriptase complex (RTC) and packaged the
N protein revealed a protein core composed of antiparallel beta genome into virion particles (Sturman et al., 1980; Hurst et al.,
sheets, hairpin extension and a hydrophobic platform which may 2013).
be implicated in RNA binding (Fan et al., 2005). The N monomer Using stable isotope labelling with amino acid in cell culture
is composed of two domains: N-terminal domain (NTD) and the (SILAC), Emmott and colleagues have identified numerous cellu-
C-terminal domain (CTD), each with a different binding mecha- lar proteins as potentially binding to the IBV N protein (Emmott
nism (Fig. 5.10). Within the NTD of the N protein, site-directed et al., 2013). Among the identified proteins, siRNA-mediated
mutagenesis has revealed Arg76 and Tyr94 to be critical for its knockdown of nucleolin, RPL19 and GSK3 has been shown to
RNA-binding activity (Tan et al., 2006). While both domains significantly inhibit IBV replication in cell culture, suggesting the
are capable of binding to the viral RNA genome in vitro, binding important functions of these host proteins during IBV infection
contributions from both domains are required to achieve optimal (Emmott et al., 2013).
RNA binding (Chang et al., 2006; Hurst et al., 2009). Previous studies have shown that the N protein of TGEV is
Additionally, the N protein is a phosphoprotein, modified at a cleaved at the late stage of infection, presumably by the activated
limited number of serine and threonine residues. Ser190, Ser192, caspase-6 and -7 during TGEV-induced apoptosis (Eléouët
Thr378, and Ser379 have been revealed as the phosphorylation et al., 2000). Similarly, the N protein of SARS-CoV has been
sites in IBV (Chen et al., 2005), and a cellular kinase is responsi- shown to be cleaved by caspases during lytic infection in Vero
ble for the phosphorylation in the N protein (Fang et al., 2013). E6 and A549 cells, but not cleaved during persistent infection
This heavy phosphorylation is thought to play a role in increasing in Caco-2 and N2a cells (Diemer et al., 2008). Cleavage of the
the N protein affinity to bind viral versus non-viral RNA (Spencer SARS-CoV N protein is mediated by caspase-6 and/or caspase-3
et al., 2008). The most conspicuous function of the N protein is and is dependent on the nuclear localization of the N protein
to bind viral RNA. While two RNA substrates were identified (Diemer et al., 2008). Interestingly, a recent study has shown

Figure 5.10 Linear and folded representations of coronavirus nucleocapsid (N) protein. Important residues for the infectious bronchitis virus
(IBV) N protein are S , S , T , S (phosphorylation sites). CTD, C-terminal domain; NLS, nuclear localization signal; NTD, N-terminal
190 192 378 379
domain; SR, serine–arginine-rich region.