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.

Keywords: CTD; N protein; NL63; NTD; coronavirus; nucleocapsid; structure.

FIG 1

Structure-guided alignment of the NTD (A) and CTD (B) of N protein. Secondary structures are indicated above the alignment. The regions of highest sequence conservation are highlighted. The residues which involvement in nucleic acid binding was tested by mutagenesis in this study are marked with asterisks. TT, turn.

FIG 2

Crystal structure of the NTD of HCoV-NL63 nucleocapsid protein. (A) Overall structure of the NTD monomer. β-Strands are depicted in pink, helices in red, and loops in blue. (B) Topology of the NTD. Color coding is as in panel A. (C) NTD dimer in the asymmetric unit. Sulfate ions from the crystallization buffer are shown in red. (D) Overlay of the NL63 NTD structure determined in this study (blue) and structures of NTDs of other coronaviruses, determined previously: infectious bronchitis virus (PDB code 2BXX; salmon), murine hepatitis virus (PDB code 3HD4; red), and severe acute respiratory syndrome coronavirus (PDB code 2OFZ; pink). The additional helical region characteristic of the NL63 NTD is highlighted in green.

FIG 3

Model of RNA interaction with the NL63 NTD. The protein model is shown in gray. Interacting residues are highlighted blue (His77 is located at the opposite side of the molecule and not visible in this view). The RNA backbone is shown in red, and each base is shown in yellow. Hydrogen bonds are depicted as black dotted lines. The inset shows the overall orientation of the RNA-binding site depicted in the main panel.

FIG 4

Crystal structure of the CTD of HCoV-NL63 nucleocapsid protein. (A) Overall structure of the CTD monomer. β-Strands are depicted in pink, helices in red, and loops in blue. (B) Topology of the CTD. Color coding is as in panel A. (C) CTD protein dimer as found in the asymmetric unit. (D) Overlay of the NL63 CTD dimer structure determined in this study (yellow) and structures of CTD dimers from other coronaviruses, determined previously: SARS-CoV (PDB code 2CJR; pink) and IBV (PDB code 2CA1; salmon).