The Nucleocapsid Protein of Human Coronavirus NL63

<div><p>Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the <i>Coronavirinae</i> subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.</p></div

Localization of the N protein in cells infected with HCoV-NL63.

<p>Three culture systems were used: 293T_ACE2⁺ cells, LLC-MK2 cells and fully differentiated human airway epithelial cultures. Single confocal planes are presented. Blue color denotes nuclei, while green represents localization of the HCoV-NL63 nucleocapsid protein. Top image in each set: scale bar corresponds to 40 μm; bottom image: scale bar corresponds to 5 μm.</p

References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.

<p>References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.</p

Thermodynamic parameters describing N protein and its domains.

<p>Thermodynamic parameters describing N protein and its domains.</p

Interaction of the CTD and NTD with RNA and DNA.

<p><b>(A)</b> RNA or DNA samples were pre-incubated with the protein and subsequently separated on the agarose gel. Shifts (the nucleoprotein complex does not leave the well in this case) observed in the lines containing RNA and DNA pre-incubated with the NTD suggest strong RNA-NTD and DNA-NTD interaction. <b>(B)</b> Electron microscopy images of the NTD in the absence and in the presence of RNA. Micrographs were prepared using scanning electron microscope JSM-5410.</p

N protein and CTD are able to form dimers.

<p>Molecular weight values for WT nucleocapsid protein and its domains, as determined using mass spectrometry. Experimental details are provided in the text.</p><p>N protein and CTD are able to form dimers.</p

Cell cycle analysis conducted on cells transfected with the NL63-N encoding mRNA or control, maxFP-green encoding mRNA.

<p>Analysis was performed using LLC-MK2 cells (<b>A</b>.) and 293T cells (<b>B</b>.). Nocodazole was used as a positive control, while non-viral mRNA and DMSO were used as negative controls.</p

Purified N protein of HCoV-NL63 and its domains.

<p>NL63-N protein was expressed in <i>E</i>. <i>coli</i> and purified as described in the Materials and Methods section. Purity of the protein was evaluated using SDS-PAGE analysis (Coomassie brilliant blue staining). N: complete NL63-N protein, NTD and CTD: N-terminal and C-terminal domains of the NL63-N protein, respectively. For each sample two different protein quantities were analyzed (10 μg and 20 μg). LMW Amersham GE Healthcare size marker was used, and corresponding sizes are presented on the left side of the figure.</p

DSC curves for the first heating scans for the complete NL63-N protein and its domains.

<p>NL63-N: complete NL63-N protein, NTD: N-terminal domain, CTD: C-terminal domain. Samples were suspended in 50 mM NH₄HCO₃, pH 7.8 and scanned at the rate of 1 K/min. Protein concentration was 1 mg/ml.</p

Localization of the N protein fused with the maxFP-Green protein in eukaryotic cells.

<p>293T_ACE2⁺ (<b>A</b>.) and LLC-MK2 (<b>B</b>.) cells were used. Single confocal planes are presented. Blue color denotes nuclei, while green represents localization of the HCoV-NL63 nucleocapsid protein. Top image in each set: scale bar corresponds to 40 μm; bottom image: scale bar corresponds to 10 μm. NL63-N: cells expressing N protein fused with the maxFP-Green protein; maxFP-Green: cells expressing maxFP-Green protein; Control: mock-transfected cells.</p