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Highlights: [•] Coronavirus N proteins share the same modular organization. [•] Structures of SARS-CoV N protein provide insight into nucleocapsid formation. [•] N protein binds to nucleic acid at multiple sites in a coupled-allostery manner. [•] A RNP packaging model highlighting the importance of disorder and modularity is proposed. [ABSTRACT FROM AUTHOR]

Abstract: The coronavirus (CoV) N protein oligomerizes via its carboxyl terminus. However, the oligomerization mechanism of the C-terminal domains (CTD) of CoV N proteins remains unclear. Based on the protein disorder prediction system, a comprehensive series of HCoV-229E N protein mutants with truncated CTD was generated and systematically investigated by biophysical and biochemical analyses to clarify the role of the C-terminal tail of the HCoV-229E N protein in oligomerization. These results indicate that the last C-terminal tail plays an important role in dimer–dimer association. The C-terminal tail peptide is able to interfere with the oligomerization of the CTD of HCoV-229E N protein and performs the inhibitory effect on viral titre of HCoV-229E. This study may assist the development of anti-viral drugs against HCoV. Structured summary of protein interactions: N and C-terminal tail peptide bind by cosedimentation in solution (View interaction) N and N bind by cosedimentation in solution (View Interaction: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) C-terminal tail peptide and N bind by fluorescence technology (View interaction) N and N bind by cross-linking study (View interaction) N and N bind by cross-linking study (View Interaction: 1, 2, 3, 4) [Copyright &y& Elsevier]

The N-terminal domain of nucleocapsid protein from human coronavirus OC43 (HCoV-OC43 N-NTD) mostly contains positively charged residues and has been identified as being responsible for RNA binding during ribonucleocapsid formation in the coronavirus. In this study, the crystallization and preliminary crystallographic analysis of HCoV-OC43 N-NTD (amino acids 58-195) with a molecular weight of 20 kDa are reported. HCoV-OC43 N-NTD was crystallized at 293 K using PEG 1500 as a precipitant and a 99.9% complete native data set was collected to 1.7 Å resolution at 100 K with an overall Rmerge of 5.0%. The crystals belonged to the hexagonal space group P65, with unit-cell parameters a = 81.57, c = 42.87 Å. Solvent-content calculations suggest that there is likely to be one subunit of N-NTD in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Abstract: RNA recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (IBV), a highly contagious avian pathogen. We have demonstrated that RNA recombination can give rise to a new viral population, supported by the finding that most isolated Taiwanese (TW) IBVs, similar to Chinese (CH) IBVs, exhibit a genetic rearrangement with the American (US) IBV at the 5’ end of the nucleocapsid (N) gene. Here, we further show that positive selection has occurred at two sites within the putative crossover region of the N-terminal domain (NTD) of the TW IBV N protein. Based on the crystal structure of the NTD, the stereographic positions of both predicted selected sites do not fall close to the RNA-binding groove. Surprisingly, converting either of the two residues to the amino acid present in most CH IBVs resulted in significantly reduced affinity of the N protein for the synthetic RNA repeats of the viral transcriptional regulatory sequence. These results suggest that modulating the amino acid residue at either selected site may alter the conformation of the N protein and affect the viral RNA–N interaction. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNA-binding capacity of the N protein. [Copyright &y& Elsevier]

Coronaviruses(CoVs) cause numerous diseases, including MiddleEast respiratory syndrome and severe acute respiratory syndrome, generatingsignificant health-related and economic consequences. CoVs encodethe nucleocapsid (N) protein, a major structural protein that playsmultiple roles in the virus replication cycle and forms a ribonucleoproteincomplex with the viral RNA through the N protein’s N-terminaldomain (N-NTD). Using human CoV-OC43 (HCoV-OC43) as a model for CoV,we present the 3D structure of HCoV-OC43 N-NTD complexed with ribonucleoside5′-monophosphates to identify a distinct ribonucleotide-bindingpocket. By targeting this pocket, we identified and developed a newcoronavirus N protein inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)(N,N-dimethylamino)acetamide hydrochloride(PJ34), using virtual screening; this inhibitor reduced the N protein’sRNA-binding affinity and hindered viral replication. We also determinedthe crystal structure of the N-NTD–PJ34 complex. On the basisof these findings, we propose guidelines for developing new N protein-basedantiviral agents that target CoVs. [ABSTRACT FROM AUTHOR]

Abstract: Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication. [Copyright &y& Elsevier]

Abstract: Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection. [Copyright &y& Elsevier]