Structural Characterization of Human Coronavirus NL63 N Protein

Abstract

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N-and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein

“…Self-association of the fulllength SARS-CoV N protein and the isolated C-terminal region (domains N2b plus spacer B/N3; residues 210-422) was first demonstrated by yeast two-hybrid analysis 31 , and the purified fulllength protein was shown to self-associate into predominantly dimers in solution 32 . The structures of the N2b domain of SARS-CoV and several related coronaviruses confirmed the obligate homodimeric structure of this domain [33][34][35][36][37][38][39] , and other work showed that the region Cterminal to this domain mediates further self-association into tetramer, hexamer, and potentially higher oligomeric forms [40][41][42] . Other studies have suggested that the protein's Nterminal region, including the RNA-binding N1b domain, can also self-associate 43,44 , highlighting the possibility that assembly of full-length N into helical filaments is mediated by cooperative interactions among several interfaces.…”

“…The structure of N 2b closely resembles that of related coronaviruses, including SARS-CoV, Infectious Bronchitis Virus (IBV), MERS-CoV, and HCoV-NL63 [33][34][35][36][37][38] . The structure is particularly similar to that of SARS-CoV, with which the N2b domain shares 96% sequence identity; only five residues differ between these proteins' N2b domains (SARS-CoV Gln268  SARS-CoV-2 A267, D291E290, H335Thr334, Gln346Asn345, and Asn350Gln349), and the structures are correspondingly similar with an overall Cα r.m.s.d of 0.44 Å across the N 2b dimer (Figure 1C).…”

“…Coronavirus N proteins possess a shared domain structure with an N-terminal RNA-binding domain and a C-terminal domain responsible for dimerization. The assembly of N protein dimers into higher-order complexes is not well understood, but likely involves cooperative interactions between the dimerization domain and other regions of the protein, plus the bound RNA [31][32][33][34][35][36][37][38][39] . Here, we present a high-resolution structure of the SARS-CoV-2 N dimerization domain, revealing an intertwined dimer similar to that of related betacoronaviruses.…”

2019 Novel Coronavirus: A mysterious threat from Wuhan, China–A current review

“…HCoV-NL63 is primarily infected children and those immunocompromised with either moderate upper respiratory symptoms such as cough, fever, and rhinorrhoea or severe LRTI, such as bronchiolitis and croup, perceived primarily in younger children (Abdul-Rasool and Fielding, 2010). It is estimated that 1-10% of the population is affected annually by cold-like symptoms of HCoV-NL63 (Szelazek et al, 2017) .…”

Molecular Characteristics, Functions, and Related Pathogenicity of MERS-CoV Proteins

“…The N-terminus of the N protein (residues 39-165) may be the RNA-binding region, and the C-terminus may be a self-binding oligomer-forming region [28]. A comparison of homology between MERS-CoV and other CoVs shows that although the N protein has low homology over the entire amino acid sequence, certain local amino acid sequences-especially the N-terminal amino acid sequence-are highly conserved.…”